Vesicular Stomatitis Virus Variants Selectively Infect and Kill Human Melanomas but Not Normal Melanocytes

Metastatic malignant melanoma remains one of the most therapeutically challenging forms of cancer. Here we test replication-competent vesicular stomatitis viruses (VSV) on 19 primary human melanoma samples and compare these infections with those of normal human melanocyte control cells. Even at a low viral concentration, we found a strong susceptibility to viral oncolysis in over 70% of melanomas. In contrast, melanocytes displayed strong resistance to virus infection and showed complete protection by interferon. Several recombinant VSVs were compared, and all infected and killed most melanomas with differences in the time course with increasing rates of melanoma infection, as follows: VSV-CT9-M51 < VSV-M51 < VSV-G/GFP < VSV-rp30. VSV-rp30 sequencing revealed 2 nonsynonymous mutations at codon positions P126 and L223, both of which appear to be required for the enhanced phenotype. VSV-rp30 showed effective targeting and infection of multiple subcutaneous and intracranial melanoma xenografts in SCID mice after tail vein virus application. Sequence analysis of mutations in the melanomas used revealed that BRAF but not NRAS gene mutation status was predictive for enhanced susceptibility to infection. In mouse melanoma models with specific induced gene mutations including mutations of the Braf, Pten, and Cdkn2a genes, viral infection correlated with the extent of malignant transformation. Similar to human melanocytes, mouse melanocytes resisted VSV-rp30 infection. This study confirms the general susceptibility of the majority of human melanoma types for VSV-mediated oncolysis.     

Corticotropin Expression by Human Melanocytes in the Skin

Highly dendritic melanocytes have been observed in rapidly proliferating seborrheic keratosis, epidermis overlying melanomas, and in melanomas. On staining for the presence of POMC with monoclonal antibody against human ACTH, the melanocytes show cytoplasmic positivity. Short term organ cultures of whole skin from the marginal zone of vitiligo patients show that 22.7% of controls and 45.5% on dark incubation in adriamycin and 87.5% exposed to a pulse of UV on adriamycin treatment show melanocytes positive for ACTH. The surrounding keratinocytes in the epidermis and in the seborrheic keratosis are negative, whereas in melanomas, isolated groups of melanocytes are positive for ACTH. These findings indicate that ACTH is expressed by the melanocytes in the G₂-phase, the activity being enhanced on UV exposure. Thus UV dependent pigmentation is associated with POMC production in human skin. From this work it is evident that the melanocyte network varies the MSH/ACTH levels in correlation with repigmentation and depigmentation in the marginal zone in vitiligo by expressing POMC locally and is related to the UV-sensitivity of the melanocytes.     

Ganglioside Expression in Melanomas From Japanese Individuals: Unusual Pattern in Two Patients With Metastatic Lesions of Acral Lentiginous Melanomas

Melanoma among Japanese is rare, and differs in its clinical and histological characteristics from that found in Caucasians. In this study, the ganglioside expression of melanoma specimens obtained from Japanese was determined and compared with previously published data on Caucasians. The ganglioside composition of 25 biopsy melanoma specimens, including 13 primary and 12 metastatic lesions, was studied using thin layer chromatography. Four gangliosides (G_M3, G_D3, G_M2, G_D2) were most commonly expressed in melanomas in Japanese. The expression of gangliosides was quite variable in both primary and metastatic melanomas as seen in previous reports. No significant differences were observed between gangliosides from primary and metastatic sites. A new type of ganglioside expression, in which G_M3 was nearly the only ganglioside (>95%), was found in metastatic tumors from two Japanese patients with acral lentiginous melanoma (ALM), which is the most common clinical and histopathological type of melanoma among Japanese but is very unusual among Caucasians. The patterns of expression were similar to those in Caucasians except for the detection of a “new” pattern.     

α-MSH and Melanogenesis in Normal Human Adult Melanocytes

Normal human skin melanocytes do not pigment consistently to α-melanocyte stimulating hormone (α-MSH) in culture. The aim of this study was to establish media conditions in which to obtain a reproducible melanogenic response to α-MSH in these cells. Twenty-five media of varying mitogen composition were examined. As previously noted by other workers, melanocyte morphology and proliferation are greatly affected by media composition. However, under the majority of media conditions that supported melanocyte survival and proliferation, cells did not respond to α-MSH with any consistent increase in dopa oxidase activity or melanin content. In only one medium condition, where basic fibroblast growth factor (bFGF) was the sole mitogen present, α-MSH induced both an increase in dopa oxidase activity (at 48%) and in melanin content (of 283%).     

The Proteolytic Potential of Normal Human Melanocytes: Comparison With Other Skin Cells and Melanoma Cell Lines

To understand the contribution of epidermal melanocytes in the proteolytic potential of human skin, we have studied melanocytes grown in a low-serum medium deprived of phorbol esters, cholera toxin, and other non-physiological supplements. We focused on the plasminogen activation system and certain matrix metalloproteinases (gelatinases). Supposing that the proteolytic activity of cells can influence binding to collagen matrix and its reorganization, we have analyzed these parameters as well. We found that human melanocytes secreted tissue-type plasminogen activator and utilised it to generate cell-bound plasmin. No urokinase-type plasminogen activator was detected in the cultures but its receptor was found in cell extracts. Both the 72 kDa and 92 kDa gelatinases were secreted by the cells and in equal amounts. In addition, melanocytes secreted the wide-spectrum proteinase inhibitor alpha-2-macroglobulin. Melanocytes cast into collagen matrices retained a rounded morphology, did not extend processes, and were unable to contract collagen lattices. As a control, these parameters were investigated in parallel in cultures of human keratinocytes, dermal fibroblasts, and two melanoma cell lines. The obtained characteristics suggest that normal human melanocytes are proteolytically active cells. This function may pertain to skin physiology and pathophysiology.     

Expression of PAX3 in Ewing's Sarcoma Family of Tumors

The Ewing's sarcoma family of tumors (ESFT) is the second most common pediatric malignancy originating in the bone and is characterized by the t(11; 22) translocation. PAX3, a member of the paired box family of genes, is expressed during embryonal development of neural crest cells and is involved in the t(2; 13) translocation found in alveolar rhabdomyosarcoma. Since ESFTs are believed to be derived from neural crest tissue, we screened a series of Ewing's sarcoma and peripheral neuroectodermal tumor cell lines and tumor specimens for expression of PAX3. We found expression of PAX3 in most, but not all, of the specimens analyzed, including cell lines and patient material.     

Production of Melanocyte‐Specific Antibodies to Human Melanosomal Proteins: Expression Patterns in Normal Human Skin and in Cutaneous Pigmented Lesions

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 1 (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.     

Melanosomal Tyrosine Transport in Normal and Pink-eyed Dilution Murine Melanocytes

Tyrosine is the endogenous substrate for melanin production within melanosomes, but the method of tyrosine transport into the melanosome has not been investigated. In the mouse, melanogenesis is disrupted by mutations in the p gene resulting in the pink-eyed dilution phenotype; it has been suggested that the p gene codes for a tyrosine transport protein. We determined that normal (melan-a) melanosome-rich granular fractions take up 10 μm [³H]tyrosine at 21.1 ± 6.1 (SEM, standard error of the mean) pmol/min/mg protein (N=7) compared with 21.3 ± 5.8 SEM pmol/min/mg protein (N=5) for pink-eyed dilution, whose plasma membrane tyrosine transport was also normal (K_m 89 μM; V_max 302 pmol/min/mg cell protein). We also demonstrated that pink-eyed dilution melanosomes are immature by virtue of their low density, high hexosaminidase activity, and lack of pigment. These data indicate that a tyrosine transport system exists in the melanosomal membrane and that the p gene does not encode a tyrosine transporter of critical importance.     

不同剂量L-酪氨酸（L—TYR）对羊驼黑素细胞增殖及酪氨酸酶mRNA表达的影响

目的：探索不同剂量的L-酪氨酸对羊驼黑素细胞增殖以及酪氨酸酶mRNA表达的影响。方法：体外培养正常羊驼皮肤黑素细胞,显微镜下观察不同剂量的L-酪氨酸（100μmol／L、200μmol／L、300μmol／L、400μmol／L）对羊驼皮肤黑素细胞增殖的影响,利用实时荧光定量PCR技术分析不同剂量L-酪氨酸对酪氨酸酶mRNA的表达量的影响。结果：研究结果显示：在培养的黑素细胞中添加了不同剂量L-酪氨酸3d后,镜检观察,黑素细胞数量相比较空白对照组有所减少,酪氨酸酶mRNA表达量增加且显著高于对照组（P〈0．05）,在200μmol／L时,酪氨酸酶mRNA表达量达到最高峰值。结论：L-酪氨酸能诱导羊驼皮肤黑素细胞酪氨酸酶mRNA表达量增高。