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N-propionyl-4-S-cysteaminylphenol (N-Pr-4-S-CAP) is a substrate for tyrosinase, which is a melanin biosynthesis enzyme and has been shown to be selectively incorporated into melanoma cells. It was found to cause selective cytotoxicity against melanocytes and melanoma cells after selective incorporation, resulting in the induction of anti-melanoma immunity. However, the underlying mechanisms for the induction of anti-melanoma immunity remain unclear. This study aimed to elucidate the cellular mechanism for the induction of anti-melanoma immunity and clarify whether N-Pr-4-S-CAP administration could be a new immunotherapeutic approach against melanoma, including local recurrence and distant metastasis. A T cell depletion assay was used for the identification of the effector cells responsible for N-Pr-4-S-CAP-mediated anti-melanoma immunity. A cross-presentation assay was carried out by using N-Pr-4-S-CAP-treated B16-OVA melanoma-loaded bone marrow-derived dendritic cells (BMDCs) and OVA-specific T cells. Administration of N-Pr-4-S-CAP induced CD8+ T cell-dependent anti-melanoma immunity and inhibited the growth of challenged B16F1 melanoma cells, indicating that the administration of N-Pr-4-S-CAP can be a prophylactic therapy against recurrence and metastasis of melanoma. Moreover, intratumoral injection of N-Pr-4-S-CAP in combination with BMDCs augmented the tumor growth inhibition when compared with administration of N-Pr-4-S-CAP alone. BMDCs cross-presented a melanoma-specific antigen to CD8+ T cells through N-Pr-4-S-CAP-mediated melanoma cell death. Combination therapy using N-Pr-4-S-CAP and BMDCs elicited a superior anti-melanoma effect. These results suggest that the administration of N-Pr-4-S-CAP could be a new strategy for the prevention of local recurrence and distant metastasis of melanoma.

A major advance of drug discovery and targeted therapy directed to cancer cells may be achieved by exploitation and immunomodulation of their unique biological property. This re-view summarizes our efforts to develop novel chemo-thermo-immuno-therapy (CTI therapy) by conjugating a melanogenesis substrate, N-propionyl cysteaminylphenol (NPrCAP: amine analog of tyrosine), with magnetite nanoparticles (MNP). In our approach, NPrCAP provides a unique drug delivery system (DDS) because of its selective incorporation into melanoma cells. It also functions as a melanoma-targeted therapeutic drug because of its production of highly reactive free radicals (melanoma-targeted chemotherapy). Moreover, utilization of MNP is a platform to develop thermo-immunotherapy because of heat shock protein (HSP) generation upon exposure to an alternating magnetic field (AMF). The feasibility of our approach was successfully shown in experimental in vivo and in vitro mouse melanoma models and in preliminary clinical trials to a limited number of advanced melanoma patients.

Malignant melanoma is one of the most malignant of all cancers. Melanoma occurs at the epidermo–dermal interface of the skin and mucosa, where small vessels and lymphatics are abundant. Consequently, from the onset of the disease, melanoma easily metastasizes to other organs throughout the body via lymphatic and blood circulation. At present, the most effective treatment method is surgical resection, and other attempted methods, such as chemotherapy, radiotherapy, immunotherapy, targeted therapy, and gene therapy, have not yet produced sufficient results. Since melanogenesis is a unique biochemical pathway that functions only in melanocytes and their neoplastic counterparts, melanoma cells, the development of drugs that target melanogenesis is a promising area of research. Melanin consists of small-molecule derivatives that are always synthesized by melanoma cells. Amelanosis reflects the macroscopic visibility of color changes (hypomelanosis). Under microscopy, melanin pigments and their precursors are present in amelanotic melanoma cells. Tumors can be easily targeted by small molecules that chemically mimic melanogenic substrates. In addition, small-molecule melanin metabolites are toxic to melanocytes and melanoma cells and can kill them. This review describes our development of chemo-thermo-immunotherapy based on the synthesis of melanogenesis-based small-molecule derivatives and conjugation to magnetite nanoparticles. We also introduce the other melanogenesis-related chemotherapy and thermal medicine approaches and discuss currently introduced targeted therapies with immune checkpoint inhibitors for unresectable/metastatic melanoma.

Melanogenesis is the biological and biochemical process of melanin and melanosome biosynthesis. Melanin is formed by enzymic reactions of tyrosinase family proteins that convert tyrosine to form brown-black eumelanin and yellow-red pheomelanin within melanosomal compartments in melanocytes, following the cascades of events interacting with a series of autocrine and paracrine signals. Fully melanized melanosomes are delivered to keratinocytes of the skin and hair. The symbiotic relation of a melanocyte and an associated pool of keratinocytes is called epidermal melanin unit (EMU). Microphthalmia-associated transcription factor (MITF) plays a vital role in melanocyte development and differentiation. MITF regulates expression of numerous pigmentation genes for promoting melanocyte differentiation, as well as fundamental genes for maintaining cell homeostasis. Diseases involving alterations of EMU show various forms of pigmentation phenotypes. This review introduces four major topics of melanogenesis cascade that include (1) melanocyte development and differentiation, (2) melanogenesis and intracellular trafficking for melanosome biosynthesis, (3) melanin pigmentation and pigment-type switching, and (4) development of a novel therapeutic approach for malignant melanoma by elucidation of melanogenesis cascade.

Three-dimensional (3D) cell culture arrays of melanoma cell spheroids were assembled to evaluate the combined effect of a melanogenesis-targeting drug, N-propionyl-4-cysteaminylphenol (NPrCAP), and heat treatment. An array-like multicellular pattern of mouse melanoma B16F1 cells in a collagen gel was established by magnetic cell labeling using a pin-holder device to exert a magnetic force. The cellular spheroids were exposed to NPrCAP and heat (42°C for 1 h) as a model of anti-cancer treatment. As a result, melanogenesis of B16F1 cells was 29-fold higher in this 3D array than in conventional two-dimensional (2D) monolayer cultures. Because the spheroid size was linearly correlated with the cell number within a spheroid, the antiproliferative effect could be evaluated in a non-destructive manner. Moreover, the half-maximal inhibitory concentration of NPrCAP coupled with heat treatment calculated from the spheroid size was 2-fold higher in the 3D array (0.30mM) than in 2D culture (0.15 mM). These results indicate that spheroid formation decreases the chemosensitivity of cancer cells, and this model would be suitable as a susceptibility assay for melanogenesis-targeting drugs. Therefore, this 3D culture model provides a better screening format to evaluate drug and physical treatments for cancer therapy than 2D formats.

Melanoma is highly metastatic, but the mechanism of melanoma cell migration is still unclear. We found that melanoma cells expressed the nicotinamide adenine dinucleotide–dependent protein deacetylase SIRT1 in the cytoplasm. Cell membrane extension and migration of melanoma cells were inhibited by SIRT1 inhibitors or SIRT1 knockdown, whereas SIRT1 activators enhanced elongation of protrusion and cellular motility. In B16F1 cells, growth factor stimulation induced lamellipodium extension, a characteristic feature at the leading edge of migrating cells, and SIRT1 was found in the lamellipodium. SIRT1 inhibitor nicotinamide (NAM) or SIRT1 small interfering RNAs suppressed the lamellipodium extension by serum or platelet-derived growth factor (PDGF). The lamellipodium formation by dominant-active Rac1 was also inhibited by NAM, a SIRT1 inhibitor. NAM inhibited the accumulation of phosphorylated Akt at the submembrane by serum or PDGF. Using fluorescence resonance energy transfer, we found that NAM impaired PDGF-dependent increase in the phosphatidylinositol-3,4,5-trisphosphate level at the leading edge. NAM inhibited the abdominal metastasis of transplanted B16F1 melanoma cells in C57BL6/J mice and improved survival. Finally, SIRT1-knockdown B16F1 cells showed significantly reduced metastasis in transplanted mice compared with that in control B16F1 cells. These results indicate that SIRT1 inhibition is a strategy to suppress metastasis of melanoma cells.

Aim: Accumulating evidence has indicated that hyperthermia using magnetite nanoparticles induces antitumor immunity. This study investigated the diversity of T-cell receptors (TCRs) in tumor-infiltrating lymphocytes after hyperthermia using magnetite nanoparticles.Materials & methods: Functionalized magnetite nanoparticles, N-propionyl-4-S-cysteaminylphenol (NPrCAP)/magnetite, were synthesized by conjugating the melanogenesis substrate NPrCAP with magnetite nanoparticles. NPrCAP/magnetite nanoparticles were injected into B16 melanomas in C57BL/6 mice, which were subjected to an alternating magnetic field for hyperthermia treatment. Results: Enlargement of the tumor-draining lymph nodes was observed after hyperthermia. The TCR repertoire was restricted in tumor-infiltrating lymphocytes, and expansion of Vβ11+ T cells was preferentially found. DNA sequences of the third complementaritydetermining regions revealed the presence of clonally expanded T cells. Conclusion: These results indicate that the T-cell response in B16 melanomas after hyperthermia is dominated by T cells directed toward a limited number of epitopes and that epitope-specific T cells frequently use a restricted TCR repertoire. Original submitted 14 May 2012; Revised submitted 30 July 2012; Published online 15 October 2012

Induced pluripotent stem (iPS) cells have the ability to differentiate into multiple cell types in the body and have an unlimited growth potential. However, iPS cell-derived melanocytes produced by existing protocols have significant limitations in developing novel strategies for regenerative medicine and cell therapies of pigmentation disorders in humans because they involve culture in media containing fetal bovine serum and nonphysiological agents. In this study, we established an in vitro approach to generate iPS cell-derived human melanocytes that have higher proliferation rates and increased melanin production compared with melanocytes prepared by previously reported approaches. Importantly, our iPS cell-derived human melanocytes are prepared in fetal bovine serum-free culture conditions that do not contain any nonphysiological agents. We designed two original methods, transferring black colonies by pipette and recovering black cell pellets from centrifuged medium, and numerous human iPS cell-derived melanocytes proliferated in gelatinous dishes coated with Matrigel after 12 days. We also succeeded in inducing melanin pigmentation in the nude mouse skin in vivo using those human iPS cell-derived melanocytes. We propose that this method using iPS cells established from T cells in the blood of normal human volunteers could be applied clinically to develop regenerative medicine and cell therapies for various forms of human pigmentation disorders.

Metastatic melanoma is resistant to conventional therapies. N-propionyl-4-S-cysteaminylphenol (NPrCAP), an N-protected sulfur-amine analog of tyrosine, is a good substrate for tyrosinase and is selectively incorporated into melanoma cells, causing cytotoxicity in vitro and in vivo. We have recently shown that intratumoral injections of NPrCAP suppress not only the growth of primary B16F1 melanoma tumors but also of secondary, re-challenged tumors. The participation of CD8(+) T cells has been suggested for the NPrCAP-mediated anti-B16 melanoma immunity. In this study, the molecular mechanism of the NPrCAP cytotoxicity and immunogenicity was examined. The phenol NPrCAP was shown to be activated by mushroom tyrosinase to the ortho-quinone N-propionyl-4-S-cysteaminyl-1,2-benzoquinone (NPrCAQ), and the structure was confirmed by reducing it to the corresponding catechol. NPrCAQ reacted rapidly with biologically relevant sulfhydryl compounds such as cysteine, glutathione and bovine serum albumin. The NPrCAQ-thiol adduct formation was proven with a model thiol N-acetylcysteine by spectroscopic methods. The production and release of NPrCAQ-protein adducts was verified in B16F1 melanoma cells in vitro and in B16F1 melanoma-bearing mice in vivo through the detection of 5-S-cysteaminyl-3-S-cysteinylcatechol after acid hydrolysis of the protein fraction. These results suggest that the phenol NPrCAP, acting as a prohapten, can be activated in melanoma cells by tyrosinase to the quinone-hapten NPrCAQ, which binds to melanosomal proteins through their cysteine residues to form possible neo-antigens, thus triggering the immunological response. NPrCAP thus represents a potential new approach to immunotherapy against metastatic melanoma.

Background N -propionyl-4- S -cysteaminylphenol (NPr-4- S -CAP) is selectively incorporated into melanoma cells and degrades them. However, it remains unclear whether NPr-4- S -CAP can induce cell death associated with the induction of host immune responses and tumor suppression in vivo. Objective To examine the molecular mechanism of NPr-4- S -CAP-mediated cytotoxicity toward melanoma cells and to test whether NPr-4- S -CAP can suppress transplanted primary and secondary B16F1 melanomas. Methods Cytotoxicity and apoptosis of melanoma cells were assessed by cell counting, flow cytometry, and detection of reactive oxygen species (ROS) and apoptotic molecules. NPr-4- S -CAP-associated host immunity was studied using a B16F1 mouse melanoma model through the application of CD4- and CD8-specific antibodies and tetramer assay. Results NPr-4- S -CAP suppressed growth of pigmented melanoma cells associated with an increase of intracellular ROS, activation of caspase 3 and DNA fragmentation, suggesting that NPr-4- S -CAP mediated ROS production, eliciting apoptosis of melanoma cells. Growth of transplanted B16F1 melanomas was inhibited after the consecutive intratumoral injections of NPr-4- S -CAP, and the tumor growth after rechallenge of B16F1 was significantly suppressed in the treated mice. This suppression occurred when the treated mice were given the anti-CD4 antibody, but not the anti-CD8 antibody. Tetramer assay demonstrated increased TYRP-2-specific CD8 + T cells in the lymph node and spleen cells prepared from NPr-4- S -CAP-treated B16F1-bearing mice. Conclusions These suggest that NPr-4- S -CAP induces apoptosis in melanoma cells through ROS production and generates CD8 + cell immunity resulting in the suppression of rechallenged B16F1 melanoma.

In order to develop melanoma-targeted in situ peptide vaccine immunotherapy, magnetite nanoparticles were conjugated with a melanogenesis substrate, N-propionyl cysteaminylphenol (NPrCAP). Magnetite nanoparticles introduced thermotherapy which caused non-apoptotic cell death and generation of heat shock protein (HSP) upon exposure to alternating magnetic field (AMF). NPrCAP was expected to develop a melanoma-targeted therapeutic drug because of its selective incorporation into melanoma cells and production of highly reactive free radicals, that result in not only oxidative stress but also apoptotic cell death by reacting with tyrosinase.

Exploitation of a specific biological property is one of the best approaches for developing novel cancer-targeted drugs. Melanogenesis substrate, N-propionyl cysteaminylphenol (NPrCAP: amine analog of tyrosine) may provide a unique drug delivery system (DDS) because of its selective incorporation into melanoma cells. It may also act as a melanoma-targeted therapeutic drug because of its production of highly reactive free radicals (melanoma-targeted chemotherapy). Utilization of magnetite nanoparticles can also be a good platform to develop thermo-immunotherapy because of heat shock protein (HSP) generation upon exposure to the alternating magnetic field (AMF). This study shows the feasibility of this approach in experimental study using in vivo and in vitro B16 melanoma cells and preliminary clinical study to a limited number of advanced melanoma patients. The therapeutic protocol against the primarily transplanted tumor with or without AMF once a day every other day for a total of three treatments not only inhibited the growth of primary transplant, but also prevented the growth of the secondary, re-challenge transplant and increased life span of the host mice. HSP70 production at the site of primary transplant and CD8 + T cell infiltration at the site of the re- challenge melanoma transplant were seen. Four patients entered in the preliminary clinical trial by following the basic outline of this animal protocol and two of them showed PR and CR. We hope to establish in situ vaccination immunotherapy for melanoma metastases by melanogenesis-targeted chemo- and thermotherapy.

How melanosomal proteins such as enzymic proteins (tyrosinase and tyrosinase-related proteins, Tyrps) and structural protein (gp100) are transported from Golgi to melanosomal compartments is not yet fully understood. A number of small GTPases have been found to be associated with melanosomes and we have identified one of them, Rab7, a regulator of vesicular transport, organelle motility, phospholipid signaling and cytosolic degradative machinery, as being involved in the transport of Tyrp1 from Golgi to stage I melanosomes. This study further characterizes the role of Rab7 as a regulator of differential sorting of melanosomal proteins in this process. Murine melanocytes were transiently transfected with a plasmid encoding either wild-type (Rab7WT), constitutively active (Rab7Q67L) or dominant-negative (Rab7N125I and Rab7T22N) Rab7. Through immunocytostaining and confocal laser scanning microscopy, we quantitatively compared the bio-distribution of melanosomal proteins between Rab7WT-expressing cells and mutant Rab7-expressing cells. We also characterized their differential elimination from melanosomal compartments by Rab7 by utilizing a proteasome inhibitor, MG132. Our findings indicate that Rab7 plays an important role in differential sorting of tyrosinase, Tyrp1 and gp100 in early melanogenesis cascade, and that it is more specifically involved with Tyrp1 than tyrosinase and gp100 in the trafficking from Golgi to melanosomes and the specific exit from the degradative process.

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.

SUMMARY Melanogenesis, i.e., synthesis of melanin and melanosomes, is a “cascade'’of event which is channelled by internal and external regulatory factors. The recognition and selection of this information and subsequent differentiation of melanogenesis (melanin type and melanosomal development) would be regulated significantly by melanosomal membrane. The melangenesis type could be switched relatively easily by UV light, hormone, and availability of tyrosinase substrate. The role of sulphydryl compounds as a regulatory factor in melangenesis type (in particular for pheomelanogenesis) may not be tied to its absolute presence or absence, but rather, to the effective concentration within the melanocyte at a given time. It is, therefore, probable that the morphogenesis of melanosomes may not follow immediately in response to melanogenesis-type changes, hence the melanocyte revealing more often mosaic forms of melanosomes in nature after exposure to non-genetic factors. The switch of melanogenesis would be significantly controlled by structural and functional availability of vesiculoglobular bodies which are encoded or associated with HMSA-5 (69 kDa) glycoprotein. This HMSA-5 protein shares a significant homology with gp75 “b-locus'’protein. However, because of our hypothesis that vesiculoglobular bodies carry post-(and pre-) tyrosinase regulatory factors involving in both pheo- and eumelanogenesis, the term “b-protein'’which focuses only on eumelanogenesis may not be applied to HMSA-5.

A 21-year-old woman with recurrent skin lesions present since December 1992 was seen at the University of Alberta hospitals in 1994. The skin lesions consisted of pruritic, painful, erythematous, “beefy” plaques on the trunk and extremities and also of vesicles, bullae, and pustules on the ankles and hands. She experienced numerous flare-ups of her eruption that required hospital admission on five separate occasions. Each episode was associated with sweats, chills, and dizziness. Headaches, arthralgias, myalgias, and generalized fatigue occurred on occasion. Previous skin biopsies suggested Wells' syndrome. Detailed investigations did not reveal any underlying etiology. The patient was a single woman who had recently moved from New Brunswick to live with her aunt and two cousins in Edmonton and to find employment. She had previously been a healthy young woman until her episodes first began in December 1992. There was a personal history of childhood atopic dermatitis, but she denied any other manifestations of atopy. Her mother had recently passed away at the age of 37 from Hodgkin's lymphoma, otherwise the family history was unremarkable. At the time of this admission she was not taking any medications; however, during the previous months in New Brunswick she had been using numerous medications including prednisone, dapsone, colchicine, and an oral contraceptive for a functional ovarian cyst. Prednisone, up to 60 mg/day, had initially appeared beneficial for her skin lesions, but later her symptoms became resistant. In addition, she suffered from many side-effects induced by chronic corticosteroid usage, inciuding biurred vision, weight gain, striae, and irreguiar menstruai periods. She had not been on corticosteroids for the past 6 weeks, and none of the other medications were of benefit. In fact, she had suffered a severe idiosyncratic reaction to dapsone consisting of high fever, abdominai pain, and abnormal liver parameters. There was no significant travel history and she denied any drug abuse or HIV risk factors, the patient was an animal lover and often played for hours at a time with her aunt's dog and two cats. By coincidence, in New Brunswick she had also owned a dog and two cats. A review of systems was unremarkable. On physical examination, she was a moderately obese young woman who appeared cushingoid, but otherwise well. She was afebrile. Examination of the skin revealed multiple vesicles, bullae, and pustules on her hands and fingers, involving both dorsal and palmar surfaces and extending to her wrists (Fig. 1). Some of the lesions had broken open forming overlying crusts. A few bullae had become hemorrhagic. There was an obvious foul-smelling odor emanating from the lesions. Multiple small excoriations were present bilaterally on the shins and two erythematous, edematous papules were seen on the abdomen. A live flea was found in her suprapubic area. No other fleas were found. The rest of the general examination was unremarkable. Pertinent laboratory investigations revealed an absolute eosinophilia of 2700/mm3 (24% eosinophilia) with a white blood count (WBC) of 11,400/mm”. Swabs of hand lesions were culture-positive for multiple aerobes and anaerobes: 3+ growth of Staphylococcus aureus, 3+ growth of Haemophilus parainfluenza, 1 + growth of Coxiella oxytoca, 2+ growth of Streptococcus viridans, and 4-i- growth of mixed anaerobes. Virologic tests were negative. Examination of a skin biopsy specimen (Fig. 2) revealed multiple flame figures in the dermis with surrounding and interspersed eosinophils. Numerous eosinophils, / lymphocytes, and histiocytes were also present around small blood vessels. There were degenerative changes of the upper epidermis with spongiosis, suggestive of early intraepidermal vesicle formation. Direct immunofluorescence on skin specimens was negative. Skin prick testing with flea antigen was positive for immediate hypersensitivity, but negative for a delayed reaction after 48 h. On further questioning, the patient admitted that she had played with a cat just prior to her admission, which, unbeknownst to her at the time, was flea-infested. Her aunt strongly denied any fleas in the home environment, despite her pets. No other household members complained (5f insect bites or skin symptoms. The patient's skin improved remarkably with hospitalization and treatment with antibiotics; no new lesions developed while in hospital and she was discharged 12 days later. She continued to experience recurrent, sporadic eruptions that resolved without corticosteroids, but was later lost to follow-up when she returned home to New Brunswick.

To clarify the mechanisms underlying declines in wound contraction caused by basic fibroblast growth factor (bFGF) and the role of autologous fibroblasts in modulating wound healing, we have examined the expression of alpha-smooth muscle actin (alpha-SMA) and apoptosis in a model of wound healing using collagen sponges with and without bFGF (1 microg) and/or fibroblasts (1 x 10(6) cells/cm(2)) applied to experimentally produced full-thickness skin wounds in rats (n=10 for each group). At 7 days postoperatively, wounds filled with a fibroblast-seeded collagen sponge (fibroblast-seeded group) displayed a greater area of collagen sponge and a smaller area of fibroblasts compared with control wounds filled with collagen sponge alone (control group). Therefore, seeding of fibroblasts in the dermal substitute might retard degradation of the collagen sponge, inhibiting fibroblast infiltration into the substitute. By day 14, wounds filled with bFGF-treated collagen sponge without fibroblast seeding (bFGF group) displayed decreased alpha-SMA expression and significantly increased apoptosis compared with other wounds. Double staining revealed that apoptosis in alpha-SMA-positive fibroblastic cells was significantly increased in the bFGF group, suggesting that bFGF treatment is a potent stimulator of myofibroblast apoptosis. Furthermore, morphometric analysis demonstrated the significant decrease in the level of wound contraction and the degree of mature collagen bundle formation in the bFGF group by day 42. The bFGF group also showed increased bFGF expression in macrophages by day 28. These results suggest that bFGF administration to an artificial dermis promotes apoptosis of alpha-SMA-positive fibroblastic cells and inhibits alpha-SMA expression in the treated wound, thus reducing wound contraction.

Tyrosine analogs are good candidates for developing melanoma chemotherapies because melanogenesis is inherently toxic and expressed uniquely in melanocytic cells. The sulfur homolog of tyrosine, 4-S-cysteaminylphenol (4-S-CAP), was shown to be a substrate of melanoma tyrosinase and can cause selective cytotoxicity of melanocytes and melanoma cells. Previously, in order to improve the adsorption of magnetite nanoparticles to target cell surfaces, and generate heat in an alternating magnetic field (AMF) for cancer hyperthermia, we produced hyperthermia using magnetite cationic liposomes (MCL) that have a positive charge at the liposomal surface. In the present study, we constructed 4-S-CAP-loaded MCL (4-S-CAP/MCL), which act as a novel modality, combining melanoma-specific chemotherapy by 4-S-CAP with intracellular hyperthermia mediated by MCL. The 4-S-CAP/MCL exerted 4-S-CAP-mediated anticancer effects on B16 melanoma cells in vitro and in vivo. Moreover, after intratumoral injection of 4-S-CAP/MCL in vivo, the melanoma nodules were heated to 45°C under an AMF. Significantly higher therapeutic effects were observed in mice treated with the combination therapy mediated by 4-S-CAP/MCL plus AMF irradiation compared with mice treated with 4-S-CAP/MCL alone (without AMF) or mice treated with hyperthermia alone (MCL + AMF irradiation). These results suggest that this novel therapeutic tool is applicable to the treatment of malignant melanoma. (Cancer Sci 2007; 98: 424–430)

Polymerase chain reaction(PCR)を用いると帯状疱疹患者の末梢血リンパ球からも水痘帯状疱疹ウイルス(varicella-zoster virus: VZV)核酸が検出される。本研究では，札幌医大附属病院皮膚科に入院しビダラビン(アラセナ®A)600mg/日を5日間投与された帯状疱疹患者10例よりリンパ球を調製し，PCRによって末梢血リンパ球のVZV gene 29およびgene 62のDNAおよびRNAの検出を行った。検討した10例のうち基礎疾患のある重症帯状疱疹の3例と中等症の1例は補体結合反応値が128倍と高値を示し，さらにPCRによりVZV DNAおよびRNAが検出された。しかし，これら4症例とも入院7～10日後(第14病日)には末梢血リンパ球からウイルス核酸は検出されなかった。他の中等症の1例と軽症5例の末梢血リンパ球からはVZV gene 29あるいは62のDNAとRNAは検出されなかった。患部皮疹が潰瘍化した重症の1例を除き，いずれの症例も3週間以内に上皮化し2～4週間後の帯状疱疹関連神経痛はVASスコアで0/5あるいは1/5まで低下した。これらの結果より，重症例ではウイルス抗体価が上昇し末梢血リンパ球にVZV遺伝子が検出され少なくとも一部のウイルス遺伝子が発現しているが，ビダラビン600mg/日，5日間の投与によって発症2週間後には末梢血よりウイルスが駆逐されると考えられた。

Psoralen plus ultraviolet A (PUVA) photochemotherapy is widely used for the therapy of mycosis fungoides (MF). Clinical progression of MF is often associated with an increase in the size of tumour cells known as transformation. We report two patients with CD30+ large cell transformation that appeared after low-dose PUVA therapy for MF. Clinical data, histopathology, immunohistopathology and T-cell receptor gene rearrangement were studied. Nodules consisted of atypical large cells that expressed CD30. Monoclonal rearrangement of T-cell receptors was observed in one case. Low-dose PUVA therapy may be associated with CD30+ large cell transformation in patients with MF.

Patients with familial atypical multiple mole and melanoma (FAMMM) [so-called familial dysplastic naevus syndrome (FDNS)] have a high risk for the development of malignant melanoma. The underlying gene defect has an autosomal dominant inheritance with variable expression and incomplete penetrance. Fibroblasts derived from FAMMM patients have high sensitivity to UVC and mutagens, e.g. 4-nitroquinoline-1-oxide. We were interested in identifying how the combination of inherent sensitivity to UV light and abnormal melanin synthesis interacts in the development of melanoma in FAMMM patients. Intermediates of melanin synthesis produce free radicals that are toxic to cells. Atypical moles (dysplastic naevi) are engaged in the biosynthesis of abnormal melanin pigments. This study examined whether there was any abnormal melanin pigmentation or cell damage after the ectopic expression of tyrosinase in fibroblasts from FAMMM patients when compared with fibroblasts from normal subjects. Fibroblasts from FAMMM patients (3012T and 3072T) were associated with a higher sensitivity than normal human fibroblasts to the toxicity of UVB. When cells were infected with tyrosinase-expressing adenovirus (Ad-HT) and irradiated with UVB, FAMMM fibroblasts showed higher tyrosinase activity, produced more melanin pigments and were degraded more significantly than normal human fibroblasts. Western blot analysis revealed that Ad-HT-infected 3072T produced a larger amount of tyrosinase protein than did Ad-HT-infected normal fibroblasts after UVB irradiation. Our findings suggest: (1) that FAMMM fibroblasts have an unknown machinery which enhances tyrosinase expression by UVB irradiation; and (2) that the resulting increase in melanin synthesis affects the cytotoxicity of UVB to FAMMM fibroblasts. All of these processes may be involved in the genomic instability and development of melanoma in FAMMM patients.

The synthesis of melanin intermediates through tyrosinase (TYR) involves the production of cytotoxic free radicals. By using recombinant adenoviruses that express TYR, tyrosinase-related protein 1 (TYRP1) or DOPAchrome tautomerase (DCT), we analyzed the biological function of these proteins with regard to melanin production and the growth of melanocytes, fibroblasts, melanoma cells and nonmelanoma cancer cells. High-level expression of TYR produced newly synthesized melanin and induced cell death in all of these cells. However, when TYRP1 or DCT was coexpressed with TYR in melanocytes and melanoma cells, TYR-mediated cell death was clearly decreased. This decrease was not observed in nonmelanocytic cells. Western blot analysis and measurement of enzyme activity revealed that the expression of TYRP1 or DCT had little effect on the amount or activity of cointroduced TYR in either the melanocytic or nonmelanocytic cells. In cells expressing both TYR and TYRP1 or TYR and DCT, the total amount of melanin and/or eumelanin increased substantially more than that in cells expressing TYR alone. On the other hand, the level of pheomelanin was similar in these three cell types. These findings suggest that TYRP1 and DCT play an important role in suppressing TYR-mediated cytotoxicity in melanocytic cells without decreasing TYR expression and/or activity. These biological activities of TYRP1 and DCT may work through the interaction with TYR in melanosomal compartment.

The quality of nascent protein folding in vivo is influenced by the microdynamics of the proteins. Excessive collisions between proteins may lead to terminal misfolding, and the frequency of protein interactions with molecular chaperones determines their folding rates. However, it is unclear how immature protein dynamics are regulated. In this study, we analyzed the diffusion of immature tyrosinase in the endoplasmic reticulum (ER) of non-pigmented cells by taking advantage of the thermal sensitivity of the tyrosinase. The diffusion of tyrosinase tagged with yellow fluorescence protein (YFP) in living cells was directly measured using fluorescent correlation spectroscopy. The diffusion of folded tyrosinase in the ER of cells treated with brefeldin A, as measured by fluorescent correlation spectroscopy, was critically affected by the expression level of tyrosinase-YFP. Under defined conditions in which random diffusional motion of folded protein was allowed, we found that the millisecond-order diffusion rate observed for folded tyrosinase almost disappeared for the misfolded molecules synthesized at a nonpermissive high temperature. This was not because of enhanced aggregation at the high temperature, as terminally misfolded tyrosinase synthesized in the absence of calnexin interactions showed comparable, albeit slightly slower, diffusion. Yet, the thermally misfolded tyrosinase was not immobilized when measured by fluorescence recovery after photobleaching. In contrast, terminally misfolded tyrosinase synthesized in cells in which alpha-glucosidases were inhibited showed extensive immobilization. Hence, we suggest that the ER represses random fluctuations of immature tyrosinase molecules while preventing their immobilization.

Background: malignant melanoma has one of the highest rates of metastasis. Unlike other solid cancers, no sensitive tumor markers or laboratory tests that can provide information of the risk of metastasis and predict the prognosis have yet been established. Objective: the study was done to establish a RT-PCR sensitive and specific enough to detect melanocyte-specific transcripts from peripheral blood cells. Methods: peripheral white blood cells were collected from 30 healthy donors and 43 melanoma patients. Melanocyte-specific tyrosinase (TYR) and tyrosinase-related protein 1 (TYRP1) were selected as targets of RT-PCR. The sensitivity of detection using SK-mel-23 melanoma cells and rates of false-positiveness using non-melanoma blood were compared between single-step PCR and nested PCR. Analysis of melanoma blood samples was carried out by the single-step PCR. Results: the nested RT-PCR amplified the TYR and TYRP1 sequences from 14 and 2 of the 30 healthy bloods, respectively. However, the single-step RT-PCR did not amplify TYR or TYRP1 sequences from any of the healthy controls. Our single-step RT-PCR detected 1.7 and 0.8 SK-mel-23 melanoma cells per ml blood for TYR and TYRP1, respectively. Overall, TYR mRNA was detected in 15 of the 43 melanoma patients (34.9%), and TYRP1 mRNA in 16 of the 43 (37.2%). The specificities of detection of TYR and TYRP1 were 83.3 and 88.9%, respectively. Conclusion: our single-step RT-PCR for TYR and TYRP1 mRNAs is more specific than the previous nested RT-PCR for TYR and applicable to detect circulating melanoma cells.

A multicenter randomized controlled study was conducted to assess the long-term efficacy and safety of cyclosporin A therapy for psoriasis using either a continuous or an intermittent regimen. Initially, both regimens consisted of 3-5 mg/kg/day administration of CyA. Once remission was obtained, CyA dose was maintained between 0.5 and 3 mg/kg/day under the continuous regimen, while under the intermittent regimen, CyA dose was tapered off and, when necessary, topical corticosteroids were used until relapse occurred. Thirty-one patients were followed for at least 48 months (mean follow-up period: 55.9+/-4.6 months): 15 received continuous therapy, and 16 received intermittent therapy. With both regimens, the PASI (Psoriasis Area and Severity Index) score was maintained at 5-12 points throughout the follow-up period. The score was decreased by more than 70% from baseline with both regimens: the responses between them were not significantly different. However, overall control of psoriasis, as assessed from the averaged PASI score, was better in the patients receiving continuous therapy. Although the overall frequency of adverse reactions was similar for the two regimens, cancer occurred in two patients on continuous therapy (gastric cancer and hepatocellular carcinoma in one patient each). We could not, however, definitely attribute the cancers in the two patients to continuous therapy itself. There was a significantly higher incidence of renal impairment in elderly patients receiving either regimen when compared with younger patients. In conclusion, CyA administered to psoriasis patients under both regimens exhibited long-term efficacy and tolerability. Despite a lower overall efficacy, it seems proper to conclude that intermittent therapy is more useful than continuous therapy due to the occurrence of malignancies with continuous therapy. Further investigation is required to determine whether intermittent therapy is really safer than continuous therapy, and, if so, how it should be designed to minimize long-term adverse reactions and achieve overall control comparable to that of continuous CyA therapy.

We have recently identified the association of Rab7 in melanosome biogenesis and proposed that Rab7 is involved in the transport of tyrosinase-related protein 1 from the trans-Golgi network to melanosomes, possibly passing through late-endosome-delineated compartments. In order to further investigate the requirement of Rab7-containing compartments for vesicular transport of tyrosinase family proteins, we expressed tyrosinase and tyrosinase-related protein by recombinant adenovirus and analyzed their localization in human amelanotic melanoma cells (SK-mel-24) in the presence or absence of a dominant-negative mutant of Rab7 (Rab7N125I). Co-infection of the recombinant adenoviruses carrying tyrosinase (Ad-HT) and TRP-1 (Ad-TRP-1) resulted in the enhancement of tyrosinase activity and melanin production compared to a single infection of Ad-HT. In the Ad-HT-infected SK-mel-24 cells many of the newly synthesized tyrosinase proteins were colocalized in lysosomal lgp85-positive granules of the entire cytoplasm, whereas in the presence of Rab7N125I the colocalization of tyrosinase and lgp85 proteins was decreased markedly in the distal area of the cytoplasm. In the Ad-TRP-1-infected SK-mel-24 cells, TRP-1, which is reported to be present exclusively in melanosomes, was detected throughout the cytoplasm, but not colocalized in prelysosomal (early endosomal) EEA-1 granules. In the presence of Rab7N125I, however, TRP-1 was retained in the EEA-1-positive granules. Our findings indicate that the dominant-negative mutant of Rab7 impairs vesicular transport of tyrosinase and TRP-1, suggesting that the transport of these melanogenic proteins from the trans-Golgi network to maturing melanosomes requires passage through endosome-delineated compartments.

Magnetic nanoparticle-mediated hyperthermia (MNHT) generates heat to a local tumor tissue of above 43°C without damaging surrounding normal tissues. By applying MNHT, a significant amount of heat-shock proteins is expressed within and around the tumor tissues, inducing tumor-specific immune responses. In vivo experiments have indicated that MNHT can induce the regression of not only a local tumor tissue exposed to heat, but also distant metastatic tumors unexposed to heat. In this article, we introduce recent progress in the application of MNHT for antitumor treatments and summarize the mechanisms and processes of its biological effects during antitumor induction by MNHT. Several clinical trials have been conducted indicating that the MNHT system may add a promising and novel approach to antitumor therapy.

Melanoma cells rarely contain mutant p53 and hardly undergo apoptosis by wild-type p53. By using recombinant adenoviruses that express p53 or p53-related p51A or p73beta, we tested their apoptotic activities in melanoma cells. Yeast functional assay revealed a mutation of p53 at the 258th codon (AAA [K] instead of GAA [E]) in one cell line, 70W, out of six human melanoma cell lines analyzed (SK-mel-23, SK-mel-24, SK-mel-118, TXM18, 70W, and G361). Adenovirus-mediated transfer of p53, p51A, and/or p73beta suppressed growth and induced apoptotic DNA fragmentation of SK-mel-23, SK-mel-118, and 70W cells. Interestingly, p51A induced DNA fragmentation in them more significantly than p53 and p73beta. By Western blotting we analyzed levels of apoptosis-related proteins in cells expressing p53 family members. Apoptotic Bax and antiapoptotic Bcl-2 were not significantly upregulated or downregulated by expression of p53, p51A, or p73beta, except for p53-expressing 70W cells, which contained a larger amount of Bax protein than LacZ-expressing cells. Activation of caspase-3 was demonstrated only in p51A-expressing SK-mel-118 cells. We show here that p51A can mediate apoptosis in both wild-type and mutant p53-expressing melanoma cells more significantly than p53 and p73beta. It is also suggested that in melanoma cells (i) cellular target protein(s) other than Bcl-2 and Bax might be responsible for induction of p51A-mediated apoptosis and (ii) caspase-3 is not always involved in the apoptosis by p53 family members.

Cytokines, in particular IL-4 and IL-5, regulate IgE synthesis and eosinophil activation in atopic dermatitis (AD). To elucidate whether the serum levels of IL-4 and IL-5 are related to the serum IgE level, eosinophilia, or clinical severity of the disease, 25 cases with AD were studied. Blood samples were isolated from two groups of donors: 1) patients with AD (n = 25); 2) non-allergic individuals (NA, n = 20) with serum IgE levels below 100 IU/ml and with blood eosinophil counts below 250/microliter. Each parameter was evaluated at least twice in AD patients at the beginning of the study and after 4, 8 or 12 weeks of treatment. IL-4 was hardly detected in AD and NA, but IL-5 was increased (> 10 pg/ml) in most cases (22/25) of AD group with 513.6 pg/ml as the mean. AD with normal serum IgE levels exhibited increased levels of IL-5, whereas AD with high serum IgE levels did not necessarily have elevated IL-5 levels. The IL-5 level tended to change in parallel with the clinical severity in each AD case, although the level itself was not correlated with the clinical severity per se. A significant decrease of IL-5 was observed in AD when the clinical severity decreased. Eosinophils also decreased along with the improvement of AD, whereas the serum level of IgE did not change during the observation period. Our results suggest that IL-5 is involved in the regulation of clinical courses of AD and that its kinetics at the serum level reflects the clinical activity of AD.

21歳,男性。中背部右側に自覚症状を欠く11×7mmのドーム状に隆起する黒色結節を認め,単純切除術を施行。病理組織学的に結節型悪性黒色腫,tumor thickness 5.0mm, level 4であった。過去10年間に当院で経験した悪性黒色腫113例の統計では,結節型黒色腫(以下NM)は若年層男性の体幹に,表在拡大型(以下SSM)は若年層女性の下腿に発症率が高かった。又,足底,体幹の非露出部では他部位と比較して男性の発症が,一方顔面,四肢の露出部では女性の発症が多かった。

疼痛·そう痒を伴う各種皮膚疾患患者34名(男性20名,女性14名,平均年齢53.9歳)を対象に0.025%カプサイシン軟膏を外用し,その有用性を検討した。各疾患ごとの有効率は,帯状疱疹後神経痛(PHN)10例:67%,尋常性乾癬10例:67%,結節性痒疹2例:59%,慢性光線過敏性皮膚炎(CAD)3例:96%,アトピー性皮膚炎3例:41%,肥厚性瘢痕のそう痒7例:66%,肥厚性瘢痕の疼痛3例:75%であった。全症例の平均有効率は68%であった。カプサイシン軟膏外用後の刺激感·灼熱感は全症例でみられたが,症状の強い症例に対しては5%テーカイン軟膏を事前塗布することで対処した。その他の副作用として外用局所の紅斑:15%,流涙:3%を認めたが,他に問題となる副作用はみられなかった。以上より,カプサイシン軟膏は帯状疱疹後神経痛に有用であることが再確認されたとともに,慢性疼痛·そう痒を伴う各種皮膚疾患に対する局所療法としても有効であると考えられた。

Assembly, target-signaling and transport of tyrosinase gene family proteins at the initial stage of melanosome biogenesis are reviewed based on our own discoveries. Melanosome biogenesis involves four stages of maturation with distinct morphological and biochemical characteristics that reflect distinct processes of the biosynthesis of structural and enzymatic proteins, subsequent structural organization and melanin deposition occurring in these particular cellular compartments. The melanosomes share many common biological properties with the lysosomes. The stage I melanosomes appear to be linked to the late endosomes. Most of melanosomal proteins are glycoproteins that should be folded or assembled correctly in the ER through interaction with calnexin, a chaperone associated with melanogenesis. These melanosomal glycoproteins are then accumulated in the trans Golgi network (TGN) and transported to the melanosomal compartment. During the formation of transport vesicles, coat proteins assemble on the cytoplasmic face of TGN to select their cargos by interacting directly or indirectly with melanosomal glycoproteins to be transported. Adapter protein-3 (AP-3) is important for intracellular transport of tyrosinase gene family proteins from TGN to melanosomes. Tyrosinase gene family proteins possess a di-leucine motif in their cytoplasmic tail, to which AP-3 appears to bind. Thus, the initial cascade of melanosome biogenesis is regulated by several factors including: 1) glycosylation of tyrosinase gene family proteins and their correct folding and assembly within ER and Golgi, and 2) supply of specific signals necessary for intracellular transport of these glycoproteins by vesicles from Golgi to melanosomes.

Background: Sun-induced malignancies (basal cell and squamous cell carcinomas) are common in oculocutaneous albinism, however, the incidence of malignant melanoma is a topic of controversy. Objective: We have reviewed the literature and report a case of a woman with oculocutaneous albinism with an amelanotic melanoma of the anterior chest wall. Results: There are only 26 previously reported cases (both case reports and African albino population studies) in 25 patients in the literature. A 27th case with immunohistochemical and ultrastructural evaluation is presented. Conclusions: It appears that melanoma, a malignancy for which sun exposure and light colouration are felt to be major risk factors, has a low incidence among a population that is both hypopigmented and often exposed to high levels of ultraviolet light. This low incidence is poorly understood and frequently disputed.

Interleukin-8 (IL-8) belongs to the CXC chemokine family. IL-8 exerts its biological activities by binding to specific cell surface receptors, CXCR-1 and CXCR-2. Both receptors bind IL-8 with high affinity but they have different affinities for MGSA/Groα and NAP-2. It has been shown that the expression of epidermal CXCR-2 is increased in psoriasis, suggesting that activation of KC mediated by CXCR-2 contributes to the characteristic epidermal changes observed in psoriasis. In order to examine the mechanism(s) by which UVB therapy is effective for several dermatoses including psoriasis, we sought to examine if UVB would modulate the expression of CXCR-1 and CXCR-2 in human keratinocytes (KC). Constitutive expression of CXCR-1 and CXCR-2 mRNA was detected by RT-PCR in normal cultured human KC. After 100 or 300 J/m2 irradiation, a decrease in CXCR-2 mRNA was detectable from 12 h after irradiation; this downregulation was observed until 48 h after irradiation. In contrast, the CXCR-1 mRNA level was unchanged. Immunohistochemical studies and flow cytometry analysis confirmed the suppressive effect of UVB on the expression of CXCR-2 protein in cultured human keratinocytes. Immunohistochemical studies on two minimal erythema doses (2MED)-exposed and 2MED-unexposed skin from healthy volunteers revealed that CXCR-2 staining occurred over the whole layer of the epidermis but at 24 h after 2MED irradiation, the positive staining of CXCR-2 was decreased. A faint CXCR-1 staining was observed in the lower part of the epidermis both in unexposed and exposed skins. Our results indicate that UVB-induced growth inhibition of KC in hyperproliferative skin disorders may, in part, be related to downregulation of CXCR-2. J. Cell. Physiol. 182:366–370, 2000. © 2000 Wiley-Liss, Inc.

Sulfur-containing tyrosine analogs such as 4-S-cysteami- nylphenol (4-S-CAP) and its N-acetyl derivative, N-acetyl-4-S-CAP, are tyrosinase substrates and can cause selective cytotoxicity or cell death of melanocytes and melanoma cells. It is not clear, however, if the cytotoxicity derives from a cytostatic or cytocidal effect. The latter can also be either apoptotic or necrotic. This paper summarizes our attempt to clarify the nature of melanocytotoxicity and cell death by using a new derivative of 4-S-CAP, N-propionyl-4-S-CAP (NPr-CAP). The i.p. administration of NPr-CAP caused marked depigmentation of black hair follicles in C57 mice. At 12h postadministration of NPr-CAP, follicular melanocytes showed histochemical and morphologic features indicative of apoptosis by TdT-mediated dUTP-biotin nick end labeling (TUNEL) staining and electron microscopy. The agarose gel electrophoresis of DNA from drug-treated melan a2 cells, an immortal melanocyte line of C57 black mice, showed the nucleosomal DNA ladder pattern. NPr-CAP caused irreversible cytotoxicity in melan a2 and the effect was inhibited by a tyrosinase inhibitor, phenylthiocarbamide. The tyrosinase-mediated cytotoxicity of NPr-CAP was further confirmed by the decreased viability of COS 7 monkey-kidney cells, which expressed a level of high tyrosinase activity through transfection of human tyrosinase cDNA. NPr-CAP, however, also transiently inhibited the proliferation of melan c cells, a control tyrosinase-negative albino melanocyte line, and vector-transfected COS 7 cells. Thus, the major process of NPr-CAP-mediated melanocytotoxicity involves cytocidal apoptosis associated with active tyrosinase. In addition, there is transient, nontyrosinase-mediated cytostatic cytotoxicity.

Extramammary Paget's disease is a slowgrowing malignant disease occurring on the anogenital area and rarely on the axilla. Recent immunohistochemical studies have shown that Paget cells differentiate to secretory cells of sweat glands. However little is known about the expression of glycosaminoglycan in Paget cells and its relation to the differentiation of sweat glands. Therefore we studied the light and electron microscopic localization of anionic sites stained with cationic gold using postembedding method. We also studied the digestibility of anionic sites with enzymes such as neuraminidase, chondroitinase ABC, and heparitinase, because anionic sites in normal eccrine and apocrine sweat glands show different susceptibility to these enzymes. Cationic gold stained 19.6±3.0% of Paget cells at pH 2.0, although most of these anionic sites were not stained at pH 7.4. Anionic sites in Paget cells were completely digested with neuraminidase, however chondroitinase ABC or heparitinase did not digest them. Enzyme susceptibility and paradoxical pH-dependency of anionic sites in Paget cells were the same as those of apocrine secretory cells and completely different from those of eccrine secretory cells, ductal cells of sweat glands or epidermal keratinocytes. Therefore, the expression of glycosaminoglycan labeled with cationic gold indicates that Paget cells differentiate into secretory cells of apocrine sweat gland.

To understand the process of expression of tyrosinase, a key enzyme of melanogenesis, we examined its maturation in the endoplasmic reticulum (ER) by using a heterogeneous expression system. When human tyrosinase cDNA was introduced into COS 7 cells, tyrosinase activity was minimally detected. Immunofluorescence study revealed that tyrosinase was immunolocalized in the nuclear rim, the reticular network, and the punctuated structures. Because a cytoplasmic tail of tyrosinase-gene family protein functions as a lysosomal targeting signal in non-melanocytic cells, and immature and/or misfolded molecules are selectively retained in the ER, the observed localization suggested the inefficient maturation in the COS 7 cells. We thus examined if supplementation of calnexin, a membrane-bound chaperone with affinity for oligosaccharide-processing intermediates containing monoglucose, could improve the process. As expected, the activity was enhanced approximately 2-fold by co-transfection of cDNA encoding calnexin. In contrast, co-transfection of the cytosolic tail-free calnexin, which inhibits calnexin function by allowing premature egress of its ligands from the ER, suppressed expression of this enhanced tyrosinase activity. When alpha-glucosidase activity, which is required for calnexin function, was inhibited by castanospermine (CST) treatment, expression of tyrosinase activity was completely abolished. To confirm the direct involvement of calnexin in tyrosinase maturation, the interaction of calnexin with tyrosinase was examined. Immunoprecipitation of calnexin from extracts of [35S]methionine labeled cells with anti-calnexin antibody revealed that the association is highest immediately after the pulse and that nascent tyrosinase is gradually dissociated upon chase. The association was completely inhibited when CST was included in the medium. Hence, we suggest that the proper folding of tyrosinase is largely dependent on its direct interaction with calnexin for the determined duration in the ER.

This study characterizes the induction of melanogenesis and the expression of tyrosinase, tyrosinase-related protein (TRP) and lysosome-associated membrane protein (LAMP) gene families in the cultured melanocyte lines of non-agouti mice with four major genetic loci, i.e. melan-a2 (black, wild type), melan-b (brown, TRP-1 mutation), melan-s (black, piebaldism mutation) and melan-c (white, tyrosinase mutation) in response to repeated exposure to ultraviolet (UV) B (5 mJ/cm2, 7 consecutive days). Electron microscopy showed that new melanogenesis was induced in melan-a2, melan-s and melan-b melanocytes. Melan-a2, melan-s and melan-b showed an almost twofold increase in tyrosinase activity and gene expression with increased synthesis of melanosomes, although melan-b showed a minimum increase in tyrosinase activity. There was a twofold upregulation of LAMP-1 mRNA but no alteration in LAMP-2 and LAMP-3 mRNA expression in melan-a2, while there was no alteration in LAMP-1 mRNA expression but increased expression of LAMP-2 and LAMP-3 mRNA in melan-s, LAMP-3 showing a higher increase. Melan-b cells showed the same gene expression of LAMP-1, LAMP-2 and LAMP-3 as that of non-UV exposed cells. All three lines, however, exhibited simultaneously cell death, melan-b reaching the highest rate of cell death (96.5%). In contrast, melan-c, which did not have any tyrosinase activity with failure of melanogenesis induction, expressed all the mRNAs of the tyrosinase and LAMP gene families, but was not associated with any significant melanocyte death. Our study indicated: (i) that melanogenesis induction and melanocyte death are two photobiological processes occurring simultaneously after repeated UVB exposure, (ii) that in response to an upregulation of tyrosinase mRNA and enzymic activity, there was a co-ordinated upregulation of the LAMP-1 gene in wild type melan-a2, while no upregulation was found in melan-s and melan-b mutants, and (iii) that UV-induced melanocyte death is related to the upregulation of the tyrosinase gene, induction of new melanogenesis and mutation of the TRP-1 gene in immortal murine melanocytes.

Chemotherapy of malignant melanoma is still a great challenge, as no effective drugs are available. The development of melanogenesis-based drugs is a promising area of research because melanogenesis is a unique biochemical pathway operating only in melanoma cells (and their normal counterparts) so that the tumour can be targeted. We have been using cysteinylphenol, a sulphur-containing analogue of tyrosine, and derivatives for that purpose. N-Acetyl-4-S-cysteaminylphenol was found to have the best antimelanoma effect in cell culture systems and in mice bearing B16 melanoma tumours. It also caused depigmentation of the skin, suggesting the possibility of use as a hypopigmenting agent. To improve the efficiency of the drug, we thought of replacing the acetyl group in N-acetyl-4-S-cysteaminylphenol with a propionyl group in the hope that increased hydrophobicity would increase the cellular uptake of the drug. N-Propionyl-4-S-cysteaminylphenol was synthesized by condensing 4-hydroxythiophenol with 2-ethyl-2-oxazoline. The drug showed both cytostatic and cytocidal effects in a human melanotic melanoma cell line. The drug was found to be a good depigmenting agent for the black hair follicles of C57 black mice when given s.c. for 14 days. A 10-day treatment with N-propionyl-4-S-cysteaminylphenol at 300 mg/kg body weight reduced the growth rate of B16 melanoma s.c. tumours in mice by 36%. The propionyl derivative was found to increase the life span of mice bearing melanoma more effectively than did the acetyl derivative.

The synthesis of 3-(E)-(2-iodovinyl)-N-acetyl-4-cysteaminylphenol (3-IV-N-ACA-SCAP) started from 3-iodophenol, which on condensation with cysteamine hydrochloride gave 3-iodo-4-cysteaminylphenol (1). Acetylation of 1 followed by selective deacetylation yielded 3-iodo-N-acetyl 4-cysteaminylphenol (3-1-N-Ac-4-SCAP; 3). The stereospecific reaction of (E)-1-trimethylsilyl-2-tri-n-butyltinethylene with 3 afforded the vinyl-TMS precursor 3-(E)-(2-trimethylsilylvinyl)-N-acetyl-4-cysteaminylphenol (3-TMS-N-Ac-4-SCAP; 4) in high yield. This TMS vinyl precursor is a useful synthon for high specific activity radioiodination. Iodination of the synthon 3-(E)-(2-trimethylsilylvinyl)-N-acetyl-4-cysteaminylphenol with iodine monochloride gave 3-IV-N-Ac-4-SCAP (5). High specific activity (no carrier added) 3-(E)-(2-[ 125 I]iodovinyl)-N-acetyl-4-cysteaminyl phenol was obtained in 85% radiochemical yield upon a 5 min exposure of a mixture of Na[ 125 I]I and 3-TMS-N-Ac-4-SCAP to 2450 MHz radiation in a microwave oven.

Vitiligo is a common idiopathic acquired or inherited disease with loss of normal melanin pigments and functioning melanocytes from otherwise healthy looking skin. Clinically, vitiligo can be grouped into several unique types (Figs. 1, 2). The localized type includes focal and segmental vitiligo. Generalized types are vitiligo vulgaris, acrofacial vitiligo, and universal vitiligo. A genetic predisposition is considered. 13,46 The leukoderma of vitiligo does not contain any functioning melanocytes. 35 Pathogenesis of vitiligo leukoderma is still unknown, but currently three major theories have been proposed. 11 One is the neural theory, that presupposes that there is a chemical mediator liberated at the nerve endings that destroys the melanocytes or inhibits the production of melanin pigments. A second theory proposes that melanocytes are destroyed either by themselves through generation of melanin precursors (or metabolites) or by surrounding keratinocytes that liberate chemicals that cause oxidative stresses. In normal melanocytes there is some defense or scavenging mechanism against these cytotoxic melanin precursors or chemicals generating oxidative stresses, but there is a defect in this defense process (mechanism) in vitiligo melanocytes (Fig. 3). 26 The third theory is the autoimmune hypothesis, which speculates that vitiligo is an autoimmune disease. Vitiligo patients develop autoantibodies against melanocytes. 9,39 In metastatic melanoma patients, depigmented macules similar or identical to vitiligo leukoderma can be developed by destruction of melanocytes through the interaction with cytolytic T-lymphocytes, which are activated by tyrosinase gene family proteins 7,56 and other melanogenesis-associated proteins. 4

Background. We studied the effect of FK506 ointment on rat skin allograft survival. Methods. Lewis rat (RT1') skin allografts were histologically evaluated on day 7 after transplantation to recipient ACI (RT1 a ) rats. We set histological gradings for rejection as follows: grade 1, intraepidermal blister formation; grade 2, incomplete epidermal separation from the dermis; and grade 3, complete epidermal separation from the epidermis. According to these histological criteria, the immunosuppressive effect of FK506 ointment was assessed. Results. Our data indicated that all recipient rats without FK506 ointment showed grade 3 rejection on day 7 after transplantation. In contrast, the allografts treated with FK506 ointment showed grade 1 rejection. Furthermore the blood concentration of FK506 was below 0.5 ng/ml, minimizing the systemically adverse effects of FK506. Conclusions. Thus, the present study suggests that the topical administration of FK506 on the skin allografts may be useful and effective in the suppression of skin allograft rejection.

Tyrosinase-related protein (TRP)-1 is one of the most abundant melanosomal glycoproteins involved in melanogenesis. This report summarizes our recent research efforts related to the biological role and biosynthesis of TRP-1 and its transport from TGN (trans-Golgi network) to the stage I melanosome. Our UV irradiation and tyrosinase and TRP-1 cDNA co-transfection studies indicated that human TRP-1 is involved in not only melanogenesis but also prevention of melanocyte death, which may occur during biosynthesis of melanin pigment in the presence of tyrosinase. Furthermore, a coordinated gene interaction was indicated between tyrosinase and TRP-1, resulting in upregulation of mRNA and protein expression of LAMP (lysosome-associated membrane protein)-1 that would directly prevent the tyrosinase-mediated programmed cell death of melanocytes. Similar to tyrosinase, however, TRP-1 appears to require a molecular chaperone, calnexin, which we have cloned recently. Our cDNA transfection study of tyrosinase with calnexin showed clearly the necessity of calnexin in order to have efficient, functional activity of melanosomal glycoprotein, especially tyrosinase. Once glycosylation is completed, TRP-1 will be transported from TGN to the stage I melanosome. At this stage, TRP-1 will have its own target signal, in particular, tyrosine-rich leucine residues in cytoplasmic tail. Our TRP-1 cDNA transfection and immunoelectron microscopy study shows that TRP-1 will be transported through small vesicles, probably non-clathrin-coated type, to large vacuoles, identical to the MPR (mannose-6-phosphate receptor)-positive, late endosomes. In this transport process a low molecular weight G-protein, rab-7, was isolated from the purified melanosomal protein on 2D-PAGE and identified by subsequent sequencing and PCR amplification. Confocal microscopy with double immunostaining and immunoelectron microscopy confirmed the co-localization of rab-7 and TRP-1 in the melanosomes with early stages of maturation (I-HI). Furthermore, this process will also be regulated by phosphatidylinositol 3-kinase (PI-3 kinase).