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Multiple factors affect skin pigmentation, including those that regulate melanocyte and/or keratinocyte function. Such factors, particularly those that operate at the level of the melanosome, are relatively well characterized in mice, but the expression and function of structural and enzymatic proteins in melanocytes in human skin are not as well known. Some years ago, we generated peptide-specific antibodies to murine melanosomal proteins that proved to be instrumental in elucidating melanocyte development and differentiation in mice, but cross-reactivity of those antibodies with the corresponding human proteins often was weak or absent. In an effort to characterize the roles of melanosomal proteins in human skin pigmentation, and to understand the underlying mechanism(s) of abnormal skin pigmentation, we have now generated polyclonal antibodies against the human melanocyte-specific markers, tyrosinase, tyrosinase-related protein (TYRP1), Dopachrome tautomerase (DCT) and Pmel17 (SILV, also known as GP100). We used these antibodies to determine the distribution and function of melanosomal proteins in normal human skin (adult and newborn) and in various cutaneous pigmented lesions, such as intradermal nevi, lentigo simplex, solar lentigines and malignant melanomas. We also examined cytokeratin expression in these same samples to assess keratinocyte distribution and function. Immunohistochemical staining reveals distinct patterns of melanocyte distribution and function in normal skin and in various types of cutaneous pigmented lesions. Those differences in the expression patterns of melanocyte markers provide important clues to the roles of melanocytes in normal and in disrupted skin pigmentation.

北里大学医学部附属病院皮膚科における過去25年間の転移性皮膚癌48症例の検討をおこなった。全例とも病理組織学的に確認され, 原発巣も明らかであるが, 間葉系腫瘍および皮膚癌からの皮膚転移は除いた。転移性皮膚癌の原発巣は肺癌, 乳癌, 胃癌の順で全体の70%を占めた。しかし, 全科の悪性腫瘍の発生頻度は1位が胃癌, 2位が結腸, 直腸, 肛門癌, 3位が気管, 気管支, 肺癌の順であり, 原発巣の頻度と皮膚転移とは必ずしも相関しなかった。皮膚への転移しやすさを求めると, 乳癌, 肺癌, 食道癌, 膵臓癌, 卵巣癌, 胃癌の順であった。なお, 頻度の高い転移性皮膚癌が発見された年齢は食道癌を除き50歳台と比較的若く, 皮膚転移から死亡にいたる期間は乳癌の25.60ヵ月を除くと3から5ヵ月で非常に短かった。転移などの悪性度を示すといわれるepidermal growth factor receptor(EGFR)が乳癌では皮膚転移巣, 原発巣ともに全例発現していた。以上から皮膚転移を起こしやすい癌と起こしにくい癌があるが, 胃癌のように胃集団検診や診断技術の向上が著しいと皮膚転移を起こす進行癌が減り, 結果として皮膚転移が減少していた。乳癌におけるEGFRの発現は皮膚転移を起こしやすい良いマーカーと思われるが, 転移性皮膚癌が先にみつかったあとで原発巣が発見されることは稀で, 治療に貢献することは皆無に等しい。他科との連携を良くして原発巣, 皮膚転移巣を総合的に解析していくことが今後の課題と思われた。

The expression of various melanogenic proteins, including tyrosinase, the tyrosinase-related proteins 1 (TRP1) and 2 (TRP2/DOPAchrome tautomerase), and the silver protein in human melanocytes was studied in six different human melanoma cell lines and compared to a mouse derived melanoma cell line. Analysis of the expression of tyrosinase, TRP1, TRP2, and the silver protein using flow cytometry revealed that in general there was a positive correlation between melanin formation and the expression of those melanogenic enzymes. Although several of the melanoma cell lines possessed significant activities of TRP2, the levels of DOPAchrome tautomerase in extracts of human cells were relatively low compared to those in murine melanocytes. Melanins derived from melanotic murine JB/MS cells, from melanotic human Ihara cells and HM-IY cells, from sepia melanin, and from C57BL/6 mouse hair were chemically analyzed. JB/MS cells, as well as Ihara cells and HM-TY cells, possessed significant amounts of 5,6-dihydroxyindole-2-carboxylic acid (DHICA) derived melanins, this being dependent on the activity of TRP2. Kinetic HPLC assays showed that 5,6-dihydroxyindole (DHI) produced during melanogenesis was metabolized quickly to melanin in pigmented KHm-1/4 cells, whereas DHI was stable in amelanotic human SK-MEL-24 cells. A melanogenic inhibitor that has been purified from SK-MEL-24 cells that suppressed oxidation of DHI in the presence or absence of tyrosinase, but had no effect on DHICA oxidation. The sum of these results suggests that the expression of melanogenic enzymes as well as the activity of a melanogenic inhibitor are critical to the production of melanin synthesis in humans.

Using antibodies that recognize either tyrosinase, tyrosinase-related protein-1 (TRP1), or tyrosinase-related protein-2 (TRP2, DOPAchrome tautomerase), the quantities of those melanogenic enzymes were analyzed in five melanoma cell lines that possess various degrees of melanin production. All cells except JB/MS-W increased melanin production four to 30 times after 4 d of melanocyte-stimulating hormone (MSH) treatment. Melanin production by JB/MS-W cells 'was always under background, with Or without MSH treatment. There was a positive correlation between quantities and synthetic rates of those melanogenic enzymes and their melanin formation or DOPAchrome tautomerase activities. The activity of a heat-resistant melanogenic inhibitory factor was also analyzed. The results showed, surprisingly, that pigmented cells showed higher levels of melanogenic inhibitors activity. Tyrosinase activity was increased dramatically whereas the level of melanogenic inhibitor was remarkably decreased following MSH treatment. Interestingly, melanogenic inhibitor derived from JB/MS-W cells suppressed not only tyrosinase but also DOPAchrome tautomerase, another enzyme functional in melanin production. These results clearly suggest that melanin production is regulated by a subtle balance between the activities of these enzymes and other factors such as the melanogenic inhibitor.

メラニン産生の際に重要な役割を果たすチロジナーゼの抑制因子であるアゼライン酸を20%含有する外用剤を作製し, 肝斑, 老人性色素斑などのメラニン色素増加疾患20例に1日2回外用を行った。臨床効果の判定には色彩色差計を用いた。20例中14例に2週ないし2ヵ月で効果が現われた。副作用としては皮膚のひりつき感, 発赤が2例で認められたが, 使用中止によりこれらの副作用は色素沈着を残さず消退した。これらの所見よりアゼライン酸はメラニン色素増加疾患に有用であると考えた。

1991年の1年間に北里大学皮膚科を受診し, 遅発性両側性太田母斑様色素斑と診断された11例について検討した。男女比は1:10で圧倒的に女子に多く, 平均発症年齢は36歳であった。家族歴を有する例はなく, 眼球や口腔内の色素沈着を認めた症例は皆無であった。他の真皮メラノサイトーシスを合併した症例は, 後に両側性伊藤母斑を併発した1例, 先行する太田母斑を合併した2例の計3例であった。真皮のメラノサイトは免疫組織化学的にチロジナーゼ, チロジナーゼ関連蛋白-1陽性を呈した。チロジナーゼの抑制因子であるアゼライン酸の外用は一部の症例で有効であり, 色調の減弱が認められた。

The inhibitory effects of magnesium ascorbyl phosphate (VC-PMG) on melanogenesis were investigated in vitro using purified tyrosinase, B16F10 murine melanoma cell extract, and KHm-1/4 human melanoma cells. VC-PMG inhibited tyrosinase activity at a concentration of more than 0.001% for the purified tyrosinase, at a concentration of more than 0.01% for the B16F10 murine melanoma cells extract, and at a concentration of 0.1% for the KHm-1/4 human melanoma cells.Percutaneous absorption of 14C-labeled VC-PMG was examined by applying creams containing 3% VC-PMG onto dermatomed human cadaver skin. After 48 hours, VC-PMG was retained in the skin at levels of less than 2% of the applied dose.The lightening effects of VC-PMG on hyperpigmentation disorders, such as ephelides, chloasma, and senile freckle, in human skin in vivo were also studied. Cream containing 10% VC-PMG was applied to the pigmented area of 34 patients' faces twice a day for three months. Measurement by a color difference meter showed that the color of those pigmentations for 26 patients was lightened due to the topical application of VC-PMG cream. VC-PMG cream was also applied to non-pigmented area of 27 out of 34 patients. VC-PMG cream was also effective in lightening on 11 out of 27 healthy skin.These results suggested that VC-PMG was absorbed by topical application, and stayed in the skin, and inhibited tyrosinase activity of melanocytes. In vivo results clearly showed that topical application of VC-PMG can be effective in lightening human skin pigmentations.

Several genes critical to the regulation of melanin production in mammals have recently been cloned and characterized. They map to the albino, brown, and slaty loci in mice, and encode proteins with similar structures and features, but with distinct catalytic capacities. The albino locus encodes tyrosinase, an enzyme with three distinct catalytic activities--tyrosine hydroxylase, 3,4-dihydroxyphenylalanine (DOPA) oxidase and DHI (5,6-dihydroxyindole) oxidase. The brown locus encodes TRP-1 (tyrosinase-related protein-1), which has the same, but greatly reduced, catalytic potential. The slaty locus encodes TRP-2, another tyrosinase related-protein, which has DOPAchrome tautomerase activity. In this study we have examined the enzymatic interactions of these proteins, and their regulation by a novel melanogenic inhibitor. We observed that tyrosinase activity is more stable in the presence of TRP-1 and/or TRP-2, but that the catalytic function of TRP-2 is not affected by the presence of TRP-1 or tyrosinase. Other factors also may influence melanogenesis and a unique melanogenic inhibitor suppresses tyrosinase and DOPAchrome tautomerase activities, but does not affect the spontaneous rate of DOPAchrome decarboxylation to DHI. The results demonstrate the catalytic functions of these proteins and how they stably interact within a melanogenic complex in the melanosome to regulate the quantity and quality of melanin synthesized by the melanocyte.

No successful therapy has been reported for reticulate acropigmentation of Kitamura, which is an autosomal dominant dermatosis. We treated a patient with 20% azelaic acid ointment. Within several weeks the pigmentation was remarkably decreased and no side effects were observed. Histologic examination revealed an increased number of dopa-positive melanocytes. These cells reacted strongly to staining with antityrosinase antibody or antityrosinase-related protein antibody. Electron microscopic findings showed many melanosomes within melanocytes, keratinocytes, and melanophages. These findings suggest that the hyperpig-mentation of reticulate acropigmentation of Kitamura is the result of an excess amount of melanin production caused by activation of melanocytes in the basal layer.

39歳, 男子。1989年3月に亀頭部に疼痛が出現した。2ヵ月後に全身倦怠感, 肘膝などの関節痛が出現した。某大学病院など数ヵ所を受診し, 膠原病, ベーチェット病などを疑われ, 入院精査されたが原因不明であった。その後陰茎に紅斑が出現し, 徐々に潰瘍化したという。1990年, 7月に陰茎の潰瘍を主訴とし当科受診した。初診時, 亀頭部に不整形の潰瘍を認めた。生検を含め精査するも, 原因不明であった。亀頭部の異常なしびれ感などを訴え続けたため, 精神的背景の問題も考慮し, その後も皮膚科外来にて精神科医と共に定期的に診察していたところ, 瞬間液体冷却スプレーを持参し, これを亀頭部に長時間使用したことを白状した。以上より異常体感を伴う心気神経症と診断した。

Deep heating or denaturation of collagen has been reported to be necessary for nonablative skin rejuvenation. The purpose of this study was to examine whether thermally damaged collagen is an indispensable factor to increase the amount of collagen in vivo. Epidermal and dermal responses to infrared light therapy using a Titan source were examined with the aim of correlating histological and clinical responses in human and amelanotic mouse skin.Ten, 20, or 30 J/cm2 infrared light were irradiated on the human subject's skin (thigh), while 5, 10, 20, or 30 J/cm2 were used on amelanotic mouse skin. Biopsies were taken and analyzed using hematoxylin and eosin (HE) and Elastica von Gieson stain.Ten or 20 J/cm2 infrared light increased the amount of both collagen and elastin in all layers of the dermis without denaturing the collagen in human skin. A higher dose of 30 J/cm2 also increased the amount of collagen and elastin, but denatured the collagen in human skin. (In addition to the thigh, 2 treatments of 10 J/cm2 infrared light improved skin toning and texture on the subject's face). In mouse skin, 5 or 10 J/cm2 remarkably increased the amount of both collagen and elastin, and of epidermal cells. Twenty or 30 J/cm increased the amount of collagen and elastin and the number of keratinocytes, but caused some vacuolated degeneration of keratinocytes. The presence of denatured collagen was not evident due to the high density of collagen.This study shows that the denaturation of collagen is not required to increase the amounts of collagen or elastin in vivo in human skin. The activation of the mitochondria as well as the denaturation of collagen may play important roles in infrared phototherapy.

Using B16 F10 murine melanoma cells and sublines generated from the JB/MS melanoma which exhibit various degrees of melanogenesis, the relationships among differentiation, tumorigenicity, and metastatic potential were examined. The effect of melanocyte-stimulating hormone (MSH), which specifically stimulates differentiation of melanocytes, was also studied. All melanoma lines tested were capable of growing as experimental pulmonary metastases but, surprisingly, the undifferentiated and amelanotic JB/MS-w cells failed to grow as primary subcutaneous tumors. JB/MS-w cells, which had few surface MSH receptors, did not respond to MSH with an increase in melanin production, unlike the other cell lines. Although in vitro treatment with MSH did not change the rates of growth of primary tumors by these cell lines, such treatment decreased the number of pulmonary metastases from B16 F10, JB/MS cells, JB/MS-b1 cells and JB/MS-w cells. Conversely, MSH treatment significantly increased the rates of pulmonary metastases from JB/MS-p cells. The expression of surface melanoma antigens, urokinase-type plasminogen activity and susceptibility to natural killer cells were examined. MSH did not significantly alter surface melanoma antigen expression, but increased the natural killer cell susceptibility of B16 F10, JB/MS and JB/MS-b1 cells, cells which possess abundant surface MSH receptors. There was an inverse correlation between differentiation (pigmentation) and proliferation in vitro, and the more pigmented melanoma cells (B16 F10, JB/MS and JB/MS-b1) expressed relatively lower levels of class-I MHC, relatively higher levels of class-II MHC and the highest metastatic capacity. These results demonstrate that MSH possesses the capacity to regulate not only melanogenesis, but also other factors critical to the metastatic growth of the cells.

Background: An inhibitory effect of ascorbic acid (AsA) on melanogenesis has been described. However, AsA is quickly oxidized and decomposed in aqueous solution and thus is not generally useful as a depigmenting agent. Objective: Our purpose was to examine the effect on pigmentation of magnesium-l-ascorbyl-2-phosphate (VC-PMG), a stable derivative of AsA. Methods: Percutaneous absorption of VC-PMG was examined in dermatomed human skin, and its effect on melanin production by mammalian tyrosinase and human melanoma cells in culture was also measured. A 10% VC-PMG cream was applied to the patients. Results: VC-PMG suppressed melanin formation by tyrosinase and melanoma cells. In situ experiments demonstrated that VC-PMG cream was absorbed into the epidermis and that 1.6% remained 48 hours after application. The lightening effect was significant in 19 of 34 patients with chloasma or senile freckles and in 3 of 25 patients with normal skin. Conclusion: VC-PMG is effective in reducing skin hyperpigmentation in some patients.

Two cases of transient bullous dermolysis of the newborn are reported. The first patient, a white boy, had normal skin at birth, but multiple blisters soon developed. The oral mucous membranes were not affected. All lesions healed within 4 months without scars but with many milia. At the age of 17 months the boy was reexamined and was found to have no blisters or milia. The second patient, a Japanese baby girl, had extensive denudation of her hands at birth. Generalized blisters and involvement of the oral mucous membrane developed. Blistering stopped within 1 1/4 months, and all lesions healed without scars. Histologically the blisters were subepidermal in both cases. Some lesional basal cells contained periodic acid-Schiff-positive inclusions. Electron microscopy revealed collagenolysis, diminution or loss of anchoring fibrils, and stellate inclusions in dilated rough endoplasmic reticulum in the keratinocytes of the lower epidermis. These stellate inclusions consisted of filamentous bundles with 25 to 33 nm cross-striations.

We investigated the effects of recombinant human gamma interferon on the induction of HLA-DR expression by two human squamous cell carcinoma, three trichilemmoma, one eccrine carcinoma, two adenocarcinoma cell lines, and cultured human keratinocytes in vitro. None of eight epithelial cell lines or keratinocytes expressed HLA-DR without gamma interferon treatment. In contrast, pure gamma interferon (500 IU/ml, 72-h treatment) induced HLA-DR expression on 1/2 squamous cell carcinoma, 3/3 trichilemmoma, 2/2 adenocarcinoma cell lines, and 4/4 keratinocyte cell lines, as determined using a fluorescence-activated cell sorter. A maxillary squamous cell carcinoma line and an eccrine carcinoma cell line failed to express HLA-DR with gamma interferon treatment; however, the growth of cells was inhibited by gamma interferon treatment. By indirect immunoperoxidase techniques, tumor cells such as Bowen's disease and squamous cell carcinoma were found to express HLA-DR. Since HLA-DR expression has been shown to be important for various immune responses, these findings suggest that gamma interferon plays important roles in various immune-related skin diseases.

Melanocyte stimulating hormone (MSH) specifically induces differentiation of mammalian melanocytes. To further define the biochemical events elicited by this stimulus, we have cloned murine melanoma cells which are either highly responsive or nonresponsive to MSH, and have examined their ultrastructural appearance, their melanogenic activities, and also their expression of tyrosinase. We have found that the basal levels of melanogenic activity in pigmented and nonpigmented cells correlate with expression of surface MSH receptors rather than with production of tyrosinase. Nonpigmented cells produce a potent, highly stable inhibitor of melanogenesis; this inhibitor acts directly on tyrosinase to dramatically and abruptly supress melanin production. This posttranslational control of tyrosinase activity may represent a critical regulatory point in mammalian pigmentation.

The relationship between melanogenesis and the expression of melanocyte stimulating hormone (MSH) receptors on the surface of melanocytes was examined using sublines generated from the melanotic JB/MS melanoma. JB/MS cells were propagated in long term culture to allow for phenotypic drift in their characteristics of differentiation, and then were cloned; the cloned cells ranged from well differentiated and pigmented to undifferentiated and amelanotic. Spontaneous and MSH-induced melanogenesis in these different lines was measured and correlated with the number of MSH receptors expressed. After 6 months of in vitro culture, the ability of the cells to respond to MSH was significantly reduced, as were the number of MSH receptors expressed; the cells had reduced pigmentation and were relatively undifferentiated histologically. Subsequently, clonally-derived pigmented cells were found to have numbers of surface MSH receptors (approximately 60,000 per cell) and levels of melanogenic activity similar to the original JB/MS cell line. However, an amelanotic clone had an even more dramatically reduced level of pigmentation which correlated with a further decrease in the expression of MSH receptors (less than 1,000 per cell) and the production of a potent melanogenic inhibitor. We also examined the responses of these various sublines to alpha, beta, and gamma-interferons and found significant heterogeneity in their abilities to respond to these cytokines. This study clearly shows that there is a direct correlation between melanogenesis and the expression of MSH receptors on the surface of melanocytes, and that melanogenic inhibitors may be critically involved in the regulation of mammalian pigmentation.

Using monoclonal antibodies, recombinant human gamma-interferon, and fluorescence-activated cell sorter, 2 human melanoma cell lines (KHm-1/4 and A101D) were examined quantitatively for HLA-DR and 97-kD melanoma-associated antigen (p97) expression throughout the cell cycle. Two-color flow cytometric analysis showed that the mean cell volume increased (KHm-1/4, 2.6 times; A101D, 3.6 times) during the progression of the cell cycle, and that fluorescence intensity of HLA-DR and p97 correlated well with cell volume, i.e., both antigens were maximally detected during the G2-M phase. The density of HLA-DR and p97 on the cell surface remained relatively constant throughout the cell cycle with the exception that cells in S phase showed a slightly lower density compared with those in G0/G1 and G2-M phases. gamma-Interferon treatment (500 IU/ml, 72 h) increased HLA-DR+ cells (KHm-1/4, 65% to 89%; A101D, 34% to 84%) and p97+ cells (KHm-1/4, 8% to 12%; A101D, 19% to 35%). Increased antigen densities were also relatively constant throughout the cell cycle as in nontreated cells. Cells treated with gamma-interferon tended to accumulate at G0/G1 phase (KHm-1/4, 21% to 37%; A101D, 17% to 53%), and had a reduced cell volume (0.82-0.95 times) throughout cell cycle. This study revealed that both melanoma cell lines showed heterogeneity in the expression of HLA-DR and p97, and that this heterogeneity was influenced, at least in part, by cell cycle and immunologic events such as gamma-interferon treatment.

The recently obtained JB/MS melanoma (induced by DMBA in C57Bl/6 mice) has been successfully established in culture, and characterization of various parameters of these cells, as they have been serially passaged in vivo and in vitro, has begun. The culture lines were initially highly dendritic and melanotic, growing slowly in vitro and extremely slowly in vivo. During serial passage in vivo and in vitro the cell lines have gradually evolved into less melanotic, but more proliferative, tumorigenic and metastatic cells. We have been able to demonstrate that the JB/MS melanoma shares the common melanoma TSTA previously reported for B16, K1735 and JB/RH melanomas, but does not cross-react with the S91 melanoma or with other non-melanoma cell lines used as specificity controls. The JB/MS cells can be induced to differentiate in vitro by alpha-melanocyte stimulating hormone, a physiologically relevant agent, and studies have been initiated to detail the level at which this induction occurs. These sublines should prove to be excellent models for study of the progression of transformed cells from non-tumorigenic to tumorigenic phenotypes, and for progression through stages of varying metastatic potential, immunogenicity and differentiation.