Search

Immunotherapy is well-practiced as one of the main adjuvant therapies for melanoma patients. Until now, many immunotherapeutic investigations have focused on improving the effector side of the antitumor response, but only a few studies have been concerned with preventing the loss of tumor-associated antigen (TAA) expression. Loss of TAA should be an important problem for the recognition of tumor cells by cytotoxic T lymphocytes. If any agents that augment the expression of melanoma antigens were found, they could improve the efficacy of immunotherapy by increasing the antigens. To detect effective chemicals, we made a fluorescent cellular reporter system for screening promising candidate chemicals. In this system, the fusion gene of the Melan-A/MART-1 promoter sequence followed by the green fluorescent protein (GFP) coding region was stably transfected into MUX human melanoma cells which are known to express little or no Melan-A/MART-1. Melan-A/MART-1 is a well-known melanoma antigen recognized by autologous cytotoxic T cells, and is a glycoprotein associated with the melanosome, the organelle in which melanin synthesis proceeds. By using this screening system, daunorubicin, doxorubicin and cytochalasin D, which enhanced the green fluorescent, were selected and then were confirmed to actually increase the expression of Melan-A/MART-1 mRNA and protein in human melanoma cells of MU89, MM96L(+) and SK-MEL-28, but also in low-antigen presenting cells such as MM96L(-), MUX, and A375. In conclusion, we have successfully established a well-functioning screening system, which will allow us to find candidate chemicals that up-regulate or maintain the melanoma antigen expression.

We have cloned and sequenced the putative human tyrosinase pseudogene, which shares more than 98% nucleotide homology with exon 4 and exon 5 of the human tyrosinase gene including their flanking introns. Because of such a high homology, both the tyrosinase gene and its pseudogene could be amplified from genomic DNA by polymerase chain reaction. The nucleotide sequences presented thus enable us to discriminate the tyrosinase gene from its related sequences and are invaluable for a gene diagnosis of oculocutaneous albinism.

Golgi-rich fraction was isolated from Harding-Passey mouse melanoma by centrifugation through the discontinuous sucrose density gradient and its properties were compared with those of the same fraction isolated from rat liver. The specific activity of UDP-galactose: N-acetylglucosamine galactosyltransferase was 35 times higher in the melanoma Golgi fraction than in the melanoma homogenate and was a half that in the rat liver Golgi fraction. The specific activities of marker enzymes for other subcellular components such as 5'-nucleotidase, acid phosphatase and glucose-6-phosphatase in the melanoma Golgi fraction were all one-third those in the melanoma homogenate. Electron micro-graphs of the negatively-stained Golgi fractions of melanoma and liver revealed the presence of a system of tubules, vesicles and plate-like center regions which are known as components of Golgi apparatus. Tyrosinase activity was found to be present in this fraction of mouse melanoma, but its specific activity was lower than that in the rough or smooth surface membrane fraction or in the melanosome fraction.

The decrease in tyrosinase activity following incubation of melanosomes isolated from mouse melanoma with dopa appeared to be related to the degree of in vitro melanization of melanosomes caused by incubation with dopa. The inactivation similarly occurred in the dopa-tyrosinase reaction system containing ascorbic aicd, although in the presence of ascorbic acid the dopa-quinone is immediately converted back to dopa and therefore melanin formation does not take place. The mode of inactivation of tyrosinase in melanosomes appeared to be similar to the reaction inactivation which is known to occur in the reaction of plant plant tyrosinase. Superoxide anion (O-2) and singlet oxygen (1O2) were estimated but undetectable during the dopa-tyrosinase reaction. Thus the inactivation of tyrosinase is not caused by the reaction products or the oxygen radicals.

A cDNA clone, pHT gamma 1, representing human tyrosinase mRNA was isolated by screening a melanoma cDNA library with a synthetic oligonucleotide complementary to a segment of the human tyrosinase cDNA, Pmel 34 [Kwon et al. (1987) Proc. nat. Acad. Sci. USA 84, 7473-7477]. However, there are a number of differences in the nucleotide sequence between two cDNAs, pHT gamma 1 and Pmel 34, particularly in the region coding for the carboxyl terminus of the enzyme (putative exon 5). We therefore cloned the genomic DNA segment carrying the exon 5 of the human tyrosinase gene by screening a human placental genomic DNA library with a cloned cDNA probe. The nucleotide sequences of human tyrosinase cDNA as well as part of its gene were determined. Mature human tyrosinase is composed of 511 amino acids with a molecular weight of 58,000. We provide evidence for the presence of at least two species of human tyrosinase mRNA generated by alternative splicing in human pigmented melanoma cells.

The culturing of normal human melanocytes from the roof of suction blisters of adult oriental volunteers was carried out. Since the epidermal roofs of blisters did not contain any fibroblasts, and since keratinocytes did not attach to the culture dishes in the presence of PMA (phorbol 12-myristate 13-acetate), many melanocytes were obtained which grew well in the presence of PMA. This method is a very simple and easy way to establish pure melanocyte cultures.

An increase in the amount of tyrosinase was demonstrated in the cultured human melanocyte after 6-day culturing with cholecalciferol (vitamin D3) by increased intensity of the immunofluorescent staining using monoclonal antibody against tyrosinase. Furthermore, the melanocytes became more dendritic as noted in those in the skin after the irradiation of ultraviolet. However, 7-dehydrocholesterol (pro-vitamin D3) or 1 alpha, 25-dihydroxy-vitamin D3 (activated vitamin D3) did not induce any such effect on the cultured human melanocytes. Since cholecalciferol is known to be photo-chemically converted by the ultraviolet irradiation from pro-vitamin D3 produced in the skin, the so-far-unknown mechanism of human skin pigmentation after the ultraviolet irradiation may be partly explained by this stimulating effect of vitamin D3 on the melanocytes.

Normal human epidermal melanocytes became swollen and more dendritic with an increase in the amount of immunoreactive tyrosinase when they were cultured for 2 days with each one of leukotriene (LT) C4, LTD4 and TXB2. Their effects were stronger than that of prostaglandin E2, about which we have previously reported. From these data we suggest that arachidonate-derived chemical mediators, especially LTC4, LTD4 and TXB2 may be responsible for the induction of post-inflammatory hyperpigmentation of the skin.