Histopathology of melanocytic lesions in goats and establishment of a melanoma cell line: a potential model for human melanoma

Melanocytic cells from white Angora goats were studied in vivo and in vitro. The histopathology of pigmented areas of skin from the most common sites of melanoma (solar-exposed areas of the ear, face, and perineum) resembled that of the epidermal melanocytes in Hutchinson's melanotic freckle in humans. Seven melanoma biopsies from 6 Angora goats showed histopathological features in common with human melanoma. A melanoma cell line, GM-1, was established in culture from a lymph node metastasis obtained from an animal that had a primary tumor excised and later developed extensive metastatic disease. GM-1 cells were mainly diploid, amelanotic, proliferated rapidly, spontaneously formed vacuolated cells, and were tumorigenic in nude mice. The species of origin of the GM-1 line was confirmed by isozyme profiles. GM-1 cultured cells and the original biopsy both expressed S-100 protein and tyrosinase antigen. Using GM-1 cells as the immunogen, a monoclonal antibody (MoAb 1F1) was derived that reacted strongly with a 116 kDa antigen in 50% of the GM-1 cells, but had little activity with goat fibroblasts (GM-F) or with human melanoma cells. GM-F, on the other hand, yielded more intense staining than GM-1 with an intermediate filament antibody (IFA), reacting with a 58 kDa antigen in both cell lines. The sensitivity of GM-1 to anticancer agents was similar to that of human melanoma cells. The pathology of caprine melanoma and its association with sun-exposed sites in relatively young animals suggest that it may be a suitable model for studying induction of melanoma by natural sunlight.     

Heterogeneity of tyrosinases isolated from rat skin, normal human skin, and human melanoma tumors

An electrophoretically homogeneous preparation of tyrosinase (Mr = 61 kDa) was isolated from rat skin. The purification procedure which consisted in chromatographic separation of Triton X-100-solubilized proteins included four main steps, namely: gel filtration, anion-exchange chromatography and two consecutive affinity chromatography steps. Isoelectrofocusing revealed the presence of 9 isoforms possessing an L-DOPA oxidase activity, of which proteins with pI of 4.26 and 4.33 were the major ones. The specific activity of the preparation was 43 nmol/min/mg of protein. Human skin epidermis was practically devoid of the L-DOPA-oxidase activity which was due not only to the absence of tyrosinase but also to the presence of a large amount of a 66 kDa protein able to inhibit the oxidation of L-DOPA to DOPA-chrom. The tyrosinase preparation from human melanoma consisted, predominantly, of two isoforms (48 and 69 kDa) which upon isoelectrofocusing displayed a high heterogeneity at pH around 3-5. The specific activity of the melanoma preparation markedly exceeded that of normal skin tyrosinase and was equal to 290 nmol/min/mg of protein.     

Tumor Suppressor p53 Down-Regulates Tissue-Specific Expression of Tyrosinase Gene in Human Melanoma Cell Lines

Tyrosinase, the key gene in melanin pigment synthesis, is tissue-specifically expressed in melanocytic cells. Expression of this gene is regulated by various hormones, carcinogens, and environmental factors. The molecular basis underlying tyrosinase gene regulation is still not clear. In this report, we present the effects of tumor suppressor p53 protein on tyrosinase gene expression and melanin synthesis in human melanoma. After stable transfection of wild type p53 expression plasmid into a highly pigmented melanoma cell line, overexpression of wt p53 suppressed the pigmentation of the melanoma cells. The loss of pigmentation was associated with the loss of endogenous tyrosinase expression at the activity and mRNA levels. In order to determine whether the p53 repression of tyrosinase mRNA involved modulation of tyrosinase promoter activity, transient transfection approaches involving p53 expression plasmid and construct containing chloramphenicol acetyl transferase (CAT) reporter gene linked to 270 bp tissue-specific tyrosinase promoter have been used. p53 specifically repressed CAT gene expression from the tyrosinase promoter and not from the Rous sarcoma virus promoter. These data suggest that in human melanoma p53 down-regulates the tissue-specific expression of tyrosinase gene and subsequent melanin synthesis.     

Down-regulation of melanin synthesis by a biphenyl derivative and its mechanism

Down-regulation of melanin synthesis is required for recovery of pigmentary disorders and it is known that direct inhibitors of tyrosinase, the key enzyme in melanin synthesis, such as hydroquinone with a phenol structure, suppress melanin synthesis. We screened several phenolic derivatives using B16 melanoma cells and found that a biphenyl derivative, 2,2'-dihydroxy-5,5'-dipropyl-biphenyl (DDB), down-regulated melanin synthesis effectively. Although DDB has a phenol structure, it did not inhibit tyrosinase in vitro, thus we examined its mechanism in detail. Western blotting revealed that the amount of tyrosinase was decreased by DDB, and pulse-chase labeling and immunoprecipitation analysis showed a decrease of mature tyrosinase and acceleration of tyrosinase degradation in its presence. These results suggest that DDB down-regulates melanin synthesis by inhibiting the maturation of tyrosinase, leading to acceleration of tyrosinase degradation.     

Decreased transforming growth factor-beta response and binding in insulin-independent teratoma-derived cell lines with increased tumorigenic properties.

The mouse C3H teratoma-derived cell line 1246 is an adipogenic cell line which stringently requires insulin to proliferate and differentiate in defined medium. From this cell line an insulin-independent cell line called 1246-3A was isolated. It was found that, in contrast to 1246 cells, 1246-3A cells had lost the ability to differentiate and became tumorigenic when injected at a density of 10(6) cells/mouse into syngeneic host C3H mice. In addition, they produce in their culture medium transforming growth factor alpha- and beta-like polypeptides which stimulate their proliferation. Highly tumorigenic insulin-independent cell lines able to give rise to tumor when injected at a density of 10(4) cells/mouse were isolated by using an in vitro-in vivo shuttle technique. The highly tumorigenic cell lines have lost the response to TGF-beta 1. The binding of TGF-beta 1 to the nontumorigenic parent cell line or to cells displaying increased tumorigenic properties was investigated. The data presented here indicate that the increased tumorigenicity is accompanied by a progressive decrease of specific binding of TGF-beta 1 to the cells. However, the decreased number of cell surface TGF-beta 1 binding sites in the highly tumorigenic cells did not correlate with an increase in the secretion of TGF-beta protein by the tumorigenic cells, as all of TGF-beta produced by the cells was in a latent form. Affinity cross-linking experiments indicated that the 1246 cell line displayed several TGF-beta cross-linked molecular species (MW 140, 92, and 70 kDa). Increase of tumorigenicity was accompanied by a marked decrease in the intensity of the cross-linked bands, particularly of the 92 and 70 kDa species.     

Abnormal acidification of melanoma cells induces tyrosinase retention in the early secretory pathway.

In tyrosinase-positive amelanotic melanoma cells, inactive tyrosinase accumulates in the endoplasmic reticulum. Based on studies described here, we propose that aberrant vacuolar proton ATPase (V-ATPase)-mediated proton transport in melanoma cells disrupts tyrosinase trafficking through the secretory pathway. Amelanotic but not melanotic melanoma cells or normal melanocytes display elevated proton export as observed by the acidification of the extracellular medium and their ability to maintain neutral intracellular pH. Tyrosinase activity and transit through the Golgi were restored by either maintaining the melanoma cells in alkaline medium (pH 7.4-7.7) or by restricting glucose uptake. The translocation of tyrosinase out of the endoplasmic reticulum and the induction of cell pigmentation in the presence of the ionophore monensin or the specific V-ATPase inhibitors concanamycin A and bafilomycin A1 supported a role for V-ATPases in this process. Because it was previously shown that V-ATPase activity is increased in solid tumors in response to an acidified environment, the appearance of hypopigmented cells in tyrosinase-positive melanoma tumors may indicate the onset of enhanced glycolysis and extracellular acidification, conditions known to favor metastatic spread and resistance to weak base chemotherapeutic drugs.     

Collagen XVII is expressed in malignant but not in benign melanocytic tumors and it can mediate antibody induced melanoma apoptosis

The 180?kDa transmembrane collagen XVII is known to anchor undifferentiated keratinocytes to the basement membrane in hemidesmosomes while constitutively shedding a 120?kDa ectodomain. Inherited mutations or auto-antibodies targeting collagen XVII cause blistering skin disease. Collagen XVII is down-regulated in mature keratinocytes but re-expressed in skin cancer. By recently detecting collagen XVII in melanocyte hyperplasia, here we tested its expression in benign and malignant melanocytic tumors using endodomain and ectodomain selective antibodies. We found the full-length collagen XVII protein in proliferating tissue melanocytes, basal keratinocytes and squamous cell carcinoma whereas resting melanocytes were negative. Furthermore, the cell-residual 60?kDa endodomain was exclusively detected in 62/79 primary and 15/18 metastatic melanomas, 8/9 melanoma cell lines, HT199 metastatic melanoma xenografts and atypical nests in 8/63 dysplastic nevi. The rest of 19 nevi including common, blue and Spitz subtypes were also negative. In line with the defective ectodomain, sequencing of COL17A1 gene revealed aberrations in the ectodomain coding region including point mutations. Collagen XVII immunoreaction-stained spindle cell melanomas, showed partly overlapping profiles with those of S100B, Melan A and HMB45. It was concentrated at vertical melanoma fronts and statistically associated with invasive phenotype. Antibody targeting the extracellular aa507-529 terminus of collagen XVII endodomain promoted apoptosis and cell adhesion, while inhibiting proliferation in HT199 cells. These results suggest that the accumulation of collagen XVII endodomain in melanocytic tumors is associated with malignant transformation to be a potential marker of malignancy and a target for antibody-induced melanoma apoptosis.     

Melanotropic activity of gamma MSH peptides in melanoma cells.

We studied the pigmentary activity of the peptides gamma 1, gamma 2 and gamma 3 melanocyte stimulating hormone (MSH), which differ in the structure of their C-termini, using hamster and mouse melanoma cell lines responsive to beta-MSH by increasing tyrosinase activity. Gamma 1-MSH alone or in combination with beta-MSH had no effect on either cell line. Gamma 2-MSH alone was biologically inactive but potentiated beta-MSH stimulation of tyrosinase activity. Gamma 3-MSH at high concentration (10 microM) induced tyrosinase activity and dendrite formation in the hamster melanoma line. When added together with beta-MSH, gamma 3-MSH partially inhibited the tyrosinase activity response to beta-MSH. Thus, gamma-MSH peptides have low intrinsic melanotropic activity in mammalian melanoma cells; the specific pigmentary responses appear to be affected by the structure of the C-terminal portion.     

Detection of Mouse Tyrosinase With a Monoclonal Antibody MAT-1 Against Human Tyrosinase

In this study we explored the possible application of MAT-1, which has been established as a monoclonal antibody against human tyrosinase, for detection of mouse tyrosinase. The MAT-1 reacted with B16 mouse melanoma cells, but not with tyrosinase-negative NIH-3T3 mouse fibroblasts. In western blot analysis of the large granule fraction (LGF) of B16 cells, MAT-1 detected a single protein of 80 kDa, whose size was close to that of human tyrosinase detected with MAT-1 in extracts of human melanocytes. Furthermore, the 80 kDa band that was detected with MAT-1 in the LGF of B16 cells was also detected by DOPA reaction. In order to confirm that the protein detected with MAT-1 is tyrosinase, a transient expression assay was carried out. When mouse tyrosinase or mouse tyrosinase-related protein 1, which shares high homology with human tyrosinase, was transiently expressed in tyrosinase-negative K1735 mouse melanoma cells by cDNA transfection, MAT-1 reacted only with the cells expressing mouse tyrosinase. These results indicate that MAT-1 specifically reacts with mouse tyrosinase.     

Monoclonal antibody against a melanosomal protein in melanotic and amelanotic human melanoma cells

BALB/c mice were immunized with tyrosinase, partially purified in two stages from a human melanoma cell line. A hybridoma was obtained which produced monoclonal antibody (MoAb 1C11) reactive with 8/10 melanoma cell lines and 10/10 primary cultures of human melanocytes, neval cells, and melanomas. Immunoreactivity correlated to a certain extent with tyrosinase activity but not with melanin content. No crossreactivity was obtained with neuroblastoma, medulloblastoma, fibroblasts, keratinocytes, lymphoid cells, or murine melanomas. Purification of the antigen directly from cell lysates with a MoAb 1C11 CNBr-Sepharose affinity column gave a green-brown protein of 56 kDa with no detectable tyrosinase activity. This protein was therefore different from 60 kDa active tyrosinase, identified by enzyme activity and Western blotting with a MoAb derived previously (MoAb 5C12). Unlike 5C12, 1C11 reactivity was not destroyed by pretreatment of the antigen with periodate. Immunogold labelling showed that the 1C11-reactive antigen was associated with melanosomes, and there was close correlation between 5C12 and 1C11 reactivity in resistance to trypsin and in staining various melanocytic cell populations. MoAb 1C11 may therefore recognise a polypeptide epitope in a molecule closely linked to melanin biosynthesis.     

Introduction of 65 kDa antigen of Mycobacterium tuberculosis to cancer cells enhances anti-tumor effect of BCG therapy

Bacillus Calmette Guerin (BCG) immunotherapy has anti-tumorigenic effects against bladder cancer. To improve the efficacy of BCG therapy, we introduced the gene encoding the 65 kDa heat shock protein (hsp) of Mycobacterium tuberculosis into a mouse malignant melanoma cell line (B16). An expression vector harboring the 65 kDa antigen gene was transfected into B16 using Lipofectamine, then expression of the antigen was confirmed by RT-PCR and Western blotting. Several cell lines expressing 65 kDa antigen were established (B16/65 kDa). We also established a control cell line transfected with the vector alone (B16/con). All cell lines (B16, B16/con, B16/65 kDa) were injected intraperitoneally into syngeneic mice with or without BCG prior immunization and the development of tumor ascites was examined. To analyze the mechanism of the anti-tumor effect, CD4 T cells or CD8 T cells were depleted in vivo by administering the corresponding monoclonal antibody. B16/65k Da expressed the 65 kDa hsp of M. tuberculosis. The tumor growth of B16/65 kDa was slightly retarded in naive mice, but significantly inhibited by BCG. The anti-tumor effect was totally abrogated in mice deficient in CD4 T cells, suggesting that CD4 T cells are involved in this process. The 65 kDa hsp of M. tuberculosis was expressed after gene transduction in a malignant melanoma cell line and significantly enhanced the anti-tumor effect of BCG immunotherapy. CD4 T cells play an important role in this anti-tumor effect.