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ASC/TMS1 is an adaptor protein activating caspase-1 that stimulates processing of proIL-1␤ and proIL-18. ASC was reported to be aberrantly methylated and silenced in human breast cancers. In our present study, ASC expression was examined in 12 melanoma cell lines by Western blot analysis and in 18 benign melanocytic nevi and 32 melanoma tissues by immunohistochemical staining. ASC expression was absent or reduced in 7 of 12 (58.3%) cell lines and in 20 of 32 (62.5%) melanoma tissues, whereas all 18 benign melanocytic nevi showed intensive ASC expression. To investigate whether ASC silencing in melanoma is involved in aberrant methylation, methylation specific PCR was carried out. Five of ten (50%) melanoma tissues exhibited methylation in CpG island of ASC companied with reduced ASC expression. Six of twelve (50%) melanoma cell lines showed aberrant methylation in the ASC gene, and 4 of the 6 (66.7%) methylation positive cell lines exhibited reduced ASC expression. We characterized methylation patterns in melanoma cell lines by using bisulfite genomic sequencing, and found that the degree of aberrant methylation correlated with the level of reductive ASC expression. Treatment with demethylating agent 5-aza-2-deoxycytidine resulted in both demethylation of the ASC gene and the upregulation of ASC expression in the methylation positive melanoma cell lines. Our study shows that ASC is downregulated in melanoma, and that its suppression is partially mediated by aberrant methylation.

Hyaluronate plays a unique role in the cancer cell microenvironment. In particular, melanoma is the tumor type in which hyaluronate and hyaluronate recognition have been most closely linked to malignancy. In this study we show that a human melanoma cell line stably transfected with hyaluronate synthase cDNA displays enhanced motility. We used a fixed erythrocyte exclusion assay to isolate subsets of the WM793 human melanoma cell line that expressed either high or low amounts of hyaluronate. A cell line with a high level of hyaluronate on its surface (WM793H) displayed significantly higher cell motility on colloidal-gold-coated coverslips than did a line with a low level (WM793L). Next, in order to directly investigate the effects of hyaluronate on melanoma cell migration, we transfected cDNA encoding mouse hyaluronate synthase HAS1 or HAS2 into the re-cloned human melanoma cell line that produced a low amount of hyaluronate (WM793L) by the lipofection method. Several clonal transfectants differentially producing hyaluronate were obtained. There was a positive correlation between total hyaluronate synthesis and formation of the pericellular hyaluronate-rich matrix. We observed an increase in the migration ability of hyaluronate cDNA (HAS1 or HAS2)-transfected cells compared with control cells on glass plates covered with colloidal gold particles. A migration-inhibition assay with anti-CD44 monoclonal antibody showed blocking of the cell motility. It is speculated that the tumor cells might migrate through a hyaluronate-rich extracellular environment when they invade nearby host tissues and that hyaluronate production by the tumor cells could increase this migration. These results suggest that hyaluronate may play a role in the aggressiveness of human melanoma cells.

These findings suggest that alterations of the tumour vessels are an important factor for the prognosis of malignant melanoma, and that suppression of h-CD and CNh1 in the blood vessels in malignant melanoma reflects a structural fragility of the vessels, leading to their easy penetration by tumour cells. Defective expression of these molecules is likely to be an important marker for metastatic potential and for poor prognosis of melanoma.

Cutaneous Epstein-Barr virus (EBV)-associated B-cell lymphoma (EBVBL) in non-immunocompromised patients is very rare. Here, we report a case of cutaneous EBVBL in a 72-year-old Japanese woman without any signs of immunosuppression. She showed repeated high fever and skin eruptions on the face, limbs and palms. Histological diagnosis was diffuse large B-cell lymphoma. EBV infection was detected by in situ hybridization and Southern blotting. Immunostaining for viral proteins showed the patient to be positive for latent membrane protein 1 (LMP-1) and negative for Epstein-Barr virus nuclear antigen-1 (EBNA-2), indicating that a type II latency EBV infection pattern.

Calponin h1 (CNh1) is a basic actin-binding protein that is abundantly expressed in smooth muscle cells and involved in smooth muscle contraction by inhibiting actomyosin MgATPase. In recent studies, CNh1 was noted to suppress cell proliferation and tumorigenicity in leiomyosarcoma and tumor growth in fibrosarcoma cell lines. To further investigate the function of CNh1 as a tumor suppressor, we transfected the human CNh1 gene into a v-src-transformed rat fibroblast cell line SR-3Y1. The volume of the tumors derived from one randomly selected CNh1-transfectant (C1) in nude mice was reduced to 34.1% of that from a randomly selected vector transfectant (V1). A similar tendency was observed in another independent pair (C2, V2). Pathological analysis showed a significant decrease in the number of mitotic cells in the CNh1-transfectants. Further, a marked reduction in the number of vessels in the CNh1-transfectant was observed. DNA synthesis under conditions without serum was significantly reduced in the CNh1-transfectant (C1) compared with the control transfectant (V1), while no significant difference was seen in the cellular growth in the presence of 10% serum. A slight but significant reduction in in vitro cellular motility in the CNh1-transfectant was also observed. While the suppression of growth potential and cell motility by CNh1 transfer was significant but partial, a marked reduction in vascular endothelial growth factor (VEGF) mRNA and the secretion of VEGF protein was observed in the CNh1-transfectant. These results suggest that CNh1 plays a role as tumor suppressor in SR-3Y1 mainly by decreasing VEGF expression and angiogenesis in vivo and partially through reducing cellular proliferative potential and cell motility.

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