Kazuhito Hayashibe

Deletion of specific protein kinase C subspecies in human melanoma cells.

It has been shown that tumor‐promoting phorbol ester, 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA), stimulates the proliferation of normal human melanocytes, whereas it inhibits the growth of human melanoma cell lines. The expression of protein kinase C (PKC) subspecies, the major intracellular receptors for TPA, was examined in normal melanocytes and the four melanoma cell lines HM3KO, MeWo, HMV‐1, and G361. PKC was partially purified and then separated into subspecies by column chromatography on Mono Q and hydroxyapatite successively, and finally subjected to immunoblot analysis using antibodies specific for the PKC subspecies. Of the PKC subspecies examined, δ‐, ϵ‐, and ζ‐PKC were detected in both normal melanocytes and the four melanoma cell lines. In contrast, both α‐PKC and β‐PKC were expressed in normal melanocytes, whereas either α‐PKC or β‐PKC was detected in melanoma cells. Specifically, HM3KO, MeWo, and HMV‐1 cells were shown to contain α‐PKC but not β‐PKC, while G361 cells expressed β‐PKC but not α‐PKC. The growth of these melanoma cells was suppressed by TPA treatment, and the growth of the G361 cells lacking α‐PKC was inhibited more efficiently than the other melanoma cell lines which lacked β‐PKC. It was further shown that β‐PKC was not detected in freshly isolated human primary or metastatic melanoma tissues. These results suggest that the expression of α‐PKC or β‐PKC may be altered during the malignant transformation of normal melanocytes and that loss of α‐PKC or β‐PKC may be related to the inhibitory effect of TPA on the growth of melanoma cells.

The altered expression of α‐smooth muscle actin in basal cell epithelioma and its surrounding stroma: with special reference to proliferating cell nuclear antigen expression and adenoid differentiation

Summary Altered expression of α‐smooth muscle actin (α‐SMA) is known to indicate the morphological, tumorigenic and immunological changes occurring in tumour and stromal cells. The purpose of this study was to analyse the dynamics of α‐SMA expression in human basal cell epithelioma (BCE) cells and their surrounding stromal cells, in the process of differentiation towards cutaneous appendages such as hair, sebaceous, apocrine and eccrine glands. Using anti‐α‐SMA specific monoclonal antibody (MAb), 17 of 36 BCEs (47%) were shown to express α‐SMA, despite the usual absence of α‐SMA in all eukaryotic cells except muscle cells. Solid, adenoid and sclerosing types of BCE expressed α‐SMA more frequently, and in greater amount, than cystic, keratotic and superficial types. Furthermore, the expression of α‐SMA in BCE cells significantly paralleled the expression of proliferating cell nuclear antigen (PCNA) in these cells. Thus, the altered expression of α‐SMA may reflect the growing properties of BCE cells under the specific cellular regulations for differentiation.

HLA class I antigens in Japanese patients with melanoma.

In this study, we analyzed the frequencies of human leukocyte antigen (HLA) class I alleles in 110 Japanese patients with melanoma using serological methods, and compared such frequencies with clinical parameters. As expected, frequencies of HLA allele distribution in patients with melanoma reflected the frequencies observed in the normal Japanese population. Because these are different from populations belonging to other races (e.g., white), it followed that the HLA allele distribution in melanoma patients varies among different races. This differences may have significant implications for T-cell-mediated, HLA-restricted therapeutic modalities. No significant associations between HLA and clinical parameters were noted in this study. This report may help design future clinical trials involving therapeutic approaches based on HLA-restricted mechanisms.

Effects of L‐glutamine on Tyrosinase and γ‐glutamyl Transpeptidase of B‐16 Melanoma Cells in Culture

Omission of L‐glutamine (Gln) from the culture medium decreases tyrosinase activity and increases γ‐GTP activity in cultured melanotic B‐16 melanoma cells; inclusion of this amino acid in the medium shows the reverse effects. Its absence, however, decreases the γ‐GTP activity in non‐pigmented cells like HeLa cells, fibroblasts and amelanotic melanoma cells and its presence induces a dose dependent increase in this enzyme activity in these cells. The decrease of γ‐GTP activity in the melanotic melanoma cells by Gln is attributed to increased production of dopa and its derivatives by these cells, as evidenced by our results. Our data also indicate the inhibitory effects of dopa and several other dopa derivatives known to be produced by the melanoma cells on isolated bovine kidney γ‐GTP.

Dysplastic Nevus Syndrome: Melanoma‐prone Disease

Regardless of subsequent clinical courses of patients with dysplastic nevi (DN), substantial evidence supporting DN as one of the melanoma‐prone diseases is not yet available, especially in sporadic DN, due to the lack of genetic information other than retrospective studies in clinical observation. This study aimed at the immunohistological characterization of sporadic DN distinct from common nevi (CN) and at the evaluation of the potentiality of sporadic DN for malignant transformation. We considered our results together with previous immunological and epidemiological reports.

Complete clinical remission of tumor‐stage granulomatous mycosis fungoides after treatment with PUVA, skin electron irradiation, oral etretinate and systemic interferon‐γ

in Indonesia, known as Kerik. Other ethnic terms include Kuong in Loas and Kos khyal or Koo kchall in Kampuchea. In North America, very few cases of Gua Sha are known; it was only in 1995 that Gua Sha appeared in the English literature. Wife and husband domestic abuse could be mistakenly suspected if these practices are not recognized in an adult. A careful physical exam showing linearity and symmetry of lesions is important to determine differentiating features. Gua Sha is a harmless folk remedy that should not be critically viewed by physicians, as this only alienates the patient and makes appropriate diagnosis, therapy, and follow-up less likely. Awareness and knowledge of these cultural practices will hopefully allow physicians to be more comfortable with differentiating abuse from Gua Sha.

Tissue distribution of a melanoma-associated antigen D-1 immunogenic in patients with melanoma

A human melanoma-associated antigen D-1 was recently identified by screening an expression cDNA library derived from mRNA of cultured melanoma cells with sera of melanoma patients. The aim of this study is to present in vivo expression and precise distribution of D-1 in normal tissues and benign or malignant neoplasms. By in situ hybridization, we found that the D-1 mRNA was exclusively expressed in the cytoplasms of melanoma cells, but not in keratinocytes, fibroblasts and lymphocytes adjacent to melanoma nests. Further immunohistochemical studies revealed that the expression of D-1 antigen was distributed to both the surface and cytoplasm of melanoma cells, indicating that D-1 antigen can be recognized by killer T lymphocytes or antibodies in vivo. No significant mRNA nor peptide of D-1 was detected in basal cell carcinoma, squamous cell carcinoma and other benign tumors such as melanocytic nevi and seborrheic keratosis. We also confirmed that D-1 mRNA and peptide were not expressed in normal organs by dot blot hybridization and western blot analysis, respectively. These results will assess the suitability of recombinant D-1 protein to implement active specific immunotherapy against melanoma.

Tissue Distribution of a Melanoma-Associated Antigen Immunogenic in Patients with Melanoma as Analyzed by Polyclonal Antibodies to Recombinant Peptide Antigen

This study aimed to detect in vivo expression of human melanoma‐associated antigen D‐1, which was identified by screening an expression cDNA library constructed from mRNA extracted from cultured melanoma cells with sera from patients with melanoma. The tissue distribution of D‐1 antigen was then analyzed. Murine anti‐D‐1 recombinant peptide polyclonal antibodies were raised by immunization of in vitro synthesized D‐1 peptide against Balb/c mice and applied immunohistochemically on paraffin‐embedded tissue specimens. D‐1 antigen was found to be restrictedly expressed on melanoma cells, but not on normal melanocytes, adjacent keratinocytes, fibroblasts, lymphocytes and adnexal structures of skin. The reactivities of anti‐D‐1 antibodies did not correlate with histogenesis of the lesions, their ability to produce melanin, and/or their primary or metastatic nature. There was no positive reactivity of anti‐D‐1 antibodies with other skin tumors, including squamous cell carcinoma, basal cell epithelioma, seborrheic keratosis, and nevus cell nevus. Further, cytoplasmic expression of D‐1 antigen in melanoma cells was observed only in a certain subgroup of patients with melanoma. This indicates that the cell surface expression of D‐1 peptide requires specific transporting proteins, such as HLA molecules.

The identification of strong immunogenic human malignant melanoma antigen.

黒色腫患者が,その経過中に認識し, 応答しうる黒色腫関連抗原の同定を, 培養黒色腫細胞より作成したcDNAライブラリーの患者血清によるスクリーニングで行った。同定されたcDNA clone (D-1) がコードするポリペプチドは, 約40Kdの分子量を有し, アミノ酸配列の解析によると既報告の蛋白とは相同性を示さず,新しいヒト悪性黒色腫関連抗原と思われる。

Melanoma‐associated Antigen Synthesized In vitro for Active Specific Immunotherapy

The immunogenicity of the antigen molecule is a prerequisite for active specific immunotherapy for melanoma. Since most of the melanoma‐associated antigens recognized by the murine immune system are known to be not immunogenic in man, a detection and analysis system for melanoma‐associated antigens is required to reflect in vivo immune responses in patients with melanoma. One of the promising approaches, an attempt to develop human monoclonal antibodies from B lymphocytes of patients with melanoma, has met with limited success due to the difficulties of producing large amounts of antibodies and using them in immunochemical assays, because most of them belong to the IgM class and have low affinity.