12-O-tetradecanoylphorbol-13-acetate inhibits melanoma growth by inactivation of STAT3 through protein kinase C-activated tyrosine phosphatase(s).

The growth of most melanoma cells in vitro is inhibited by the tumor-promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). In this study, the involvement of the signal transducer and activator of transcription 3 (STAT3) in the TPA-induced growth inhibition of melanoma cells was examined. The in vitro growth and DNA synthesis of five melanoma cell lines, whose STAT3 was activated (phosphorylated), was inhibited by TPA, whereas that of WM35 and WM39 cells, whose STAT3 activity was at negligible levels, was considerably slow and not affected by TPA. Blockade of STAT3 activity by small interfering RNAs suppressed the growth of WM1205Lu cells containing constitutively activated STAT3. Treatment of WM1205Lu cells with TPA decreased both the phosphorylated STAT3 and the DNA-binding activity of STAT3. Pretreatment of WM1205Lu cells with either a protein-tyrosine phosphatase inhibitor or a protein kinase C (PKC) inhibitor prevented the inhibitory effects of TPA on the level of phosphorylated STAT3. The five melanoma cell lines containing phosphorylated STAT3 commonly expressed PKCalpha, PKCdelta, and PKCepsilon. Introduction of the dominant negative mutant of one of these PKC isoforms into WM1205Lu cells inhibited the TPA-induced dephosphorylation of STAT3. A Src inhibitor attenuated the STAT3 phosphorylation in WM1205Lu cells. These results indicate that constitutively activated STAT3 is positively regulated by c-Src and negatively regulated by a PKC-activated tyrosine phosphatase(s) in melanoma cells. Because TPA did not affect c-Src activity, we conclude that the growth inhibitory effect of TPA on melanoma cells is mediated through inactivation of STAT3 by a PKC-activated tyrosine phosphatase(s).

The prevalence of melanocytic nevi on the soles in the Japanese population.

BACKGROUND: Acral lentiginous melanoma (ALM) is the most common type of melanoma in Japan. The association between ALM and acral nevus has not been elucidated. OBJECTIVE: To investigate the prevalence and dermatoscopic patterns of plantar melanocytic nevi on the soles in the Japanese and to evaluate the relationship between acral nevi and ALM. METHODS: All outpatients (N = 1697) and melanoma patients (N = 104) were included. We examined the number, size, and dermatoscopic images of nevi. RESULTS: In the control group, the prevalence of plantar nevi was 10.9%, and the mean size was 3.8 +/- 2.4 mm. The prevalence of nevi in patients with ALM and melanoma in situ on the soles was 8.6% and that of patients with melanoma on other sites was 14.5%. The main dermatoscopic pattern was "parallel furrow" in both groups. LIMITATIONS: This was a clinical observational study only. CONCLUSION: The number, size, and dermatoscopic patterns of nevi on the soles of patients with ALM and melanoma in situ on the soles did not differ from those of the control group.

Distinct sets of genetic alterations in melanoma.

BACKGROUND: Exposure to ultraviolet light is a major causative factor in melanoma, although the relationship between risk and exposure is complex. We hypothesized that the clinical heterogeneity is explained by genetically distinct types of melanoma with different susceptibility to ultraviolet light. METHODS: We compared genome-wide alterations in the number of copies of DNA and mutational status of BRAF and N-RAS in 126 melanomas from four groups in which the degree of exposure to ultraviolet light differs: 30 melanomas from skin with chronic sun-induced damage and 40 melanomas from skin without such damage; 36 melanomas from palms, soles, and subungual (acral) sites; and 20 mucosal melanomas. RESULTS: We found significant differences in the frequencies of regional changes in the number of copies of DNA and mutation frequencies in BRAF among the four groups of melanomas. Samples could be correctly classified into the four groups with 70 percent accuracy on the basis of the changes in the number of copies of genomic DNA. In two-way comparisons, melanomas arising on skin with signs of chronic sun-induced damage and skin without such signs could be correctly classified with 84 percent accuracy. Acral melanoma could be distinguished from mucosal melanoma with 89 percent accuracy. Eighty-one percent of melanomas on skin without chronic sun-induced damage had mutations in BRAF or N-RAS; the majority of melanomas in the other groups had mutations in neither gene. Melanomas with wild-type BRAF or N-RAS frequently had increases in the number of copies of the genes for cyclin-dependent kinase 4 (CDK4) and cyclin D1 (CCND1), downstream components of the RAS-BRAF pathway. CONCLUSIONS: The genetic alterations identified in melanomas at different sites and with different levels of sun exposure indicate that there are distinct genetic pathways in the development of melanoma and implicate CDK4 and CCND1 as independent oncogenes in melanomas without mutations in BRAF or N-RAS.

Differential expression of heat shock protein 105 in melanoma and melanocytic naevi.

The objective of this study is to assess the expression of heat shock protein 105 (HSP105) in melanoma and benign melanocytic lesions. The expression of HSP105 in 62 human melanoma samples--46 primary and 16 metastatic lesions--and 42 melanocytic naevi samples, was assessed by immunohistochemistry. Western blotting was performed on melanoma cell lines, melanoma tissues with matched normal skin and melanocytic naevi. The Mann-Whitney test was used for statistical analysis and significance was considered to be P less than 0.05. Seventy-four per cent of the primary melanoma lesions and 88% of the metastatic lesions overexpressed HSP105 by immunohistochemistry. The majority of melanocytic lesions (95%) were negative (P<0.05). Western blotting detected high expression of HSP105 in melanoma cell lines and tissues. The expression of HSP105 was related to the invasiveness of the lesions. Melanocytic naevi expressed HSP105 at a level that was similar to that of normal skin. Our results show that high expression of HSP105 is associated with malignant melanoma especially advanced and metastatic lesions. The results suggest that HSP105 analysis may be a helpful tool as a poor prognostic indicator and as a diagnostic aid in problematic lesions; in addition, melanoma can be included in the growing list of tumours overexpressing HSP105 to be targeted for potential HSP105-based therapeutic strategies.

Identification of a novel cancer-testis antigen CRT2 frequently expressed in various cancers using representational differential analysis.

PURPOSE: Cancer-testis antigens are promising targets for cancer immunotherapy. Identification of additional cancer-testis antigens with frequent expression in various cancers was attempted using representational differential analysis (RDA) and immunogenicity evaluation. EXPERIMENTAL DESIGN: cDNAs preferentially expressed in testis were enriched using RDA by subtraction between testis and normal tissues. Thirty clones showing cancer-testis-like expression based on EST database analysis were evaluated by reverse transcription-PCR. A potential antigen, CRT2, was identified and its expression was analyzed with a newly generated anti-CRT2 antibody. The immunogenicity of CRT2 was examined based on reactivity with serum immunoglobulin G (IgG) from cancer patients, using Western blot and ELISA analysis, and on in vitro induction of tumor-reactive CTLs from HLA-A24 transgenic mice and human peripheral blood lymphocytes. RESULTS: CRT2 was expressed in elongated spermatids of testis among normal tissues and in various cancer cell lines and tissues. The recombinant CRT2 protein was recognized by serum IgG from patients with various cancers in Western blot and ELISA analyses. A CRT2-derived peptide was identified as an HLA-A24-restricted T-cell epitope that induced tumor-reactive CTLs. CONCLUSION: CRT2 was identified as a new cancer-testis antigen expressed in elongated spermatids of testis and in cancer tissues (particularly melanoma) that is recognized by serum IgG from cancer patients. An HLA-A24-restricted T-cell epitope capable of inducing tumor-reactive CTLs was identified, suggesting that CRT2 may be useful for cancer diagnosis and immunotherapy.

A pilot study of human interferon beta gene therapy for patients with advanced melanoma by in vivo transduction using cationic liposomes.

BACKGROUND: Cationic liposomes containing the human interferon beta (HuIFNbeta) gene (IAB-1) was used for the clinical trial for glioma patients. HuIFNbeta gene therapy showed much higher anti-tumor activity compared with the administration of HuIFNbeta protein for melanoma. These results suggest that HuIFNbeta gene therapy is an attractive strategy for the treatment of melanoma. METHODS: Stage IV or III melanoma patients with cutaneous or subcutaneous metastatic lesions were enrolled in this pilot study. IAB-1 was dissolved by sterile PBS at a concentration of 30 microg DNA/ml and was injected into cutaneous or subcutaneous metastatic nodules three times a week for 2 weeks and the effect on the injected and non-injected metastatic lesions was evaluated. RESULTS: Clinical responses were as follows (five patients): mixed response (MR) and no change in each one patient, and progressive disease in three patients. In the MR patient, the IAB-1 injected lesion disappeared clinically and histopathologically and one-half of IAB-1 non-injected skin metastases were transiently inflamed and mostly regressed. In the responded non-injected lesions of this patient, histopathologically, infiltration of CD4 positive T cells was observed around the melanoma cells in the dermis, which expressed the HLA-Class II antigen. Adverse events due to this gene therapy were not recognized in any of the patients. CONCLUSIONS: The efficacy of this gene therapy was generally insufficient; however, some immunological responses were recognized in one patient. No adverse events were observed. HuIFNbeta gene therapy could be an attractive strategy for treatment of a variety of malignancies, including melanoma, though some modifications should be required.

Clinical relevance of serum levels of matrix metallopeptidase-2, and tissue inhibitor of metalloproteinase-1 and -2 in patients with malignant melanoma.

The interaction and/or balance between matrix metallopeptidase (MMP)-2 and tissue inhibitor of metalloproteinase (TIMP)-2 in vivo may play important roles in the process of tumor growth, invasion and metastasis of malignant melanoma. In this study, we investigated the serum levels and immunohistochemical expression of MMP-2, TIMP-1 and TIMP-2 in patients with melanoma and analyzed the correlation with clinicopathological parameters. The level of serum MMP-2 in patients was significantly higher than that of the control. Moreover, the level of MMP-2 was significantly higher than that of the control in patients who were: (i) female; (ii) pT1 and pT4; (iii) with and without lymph node (LN) metastasis; (iv) in stage I and stage IV; (v) with and without recurrence; and (v) alive and dead. The level of serum TIMP-1 in patients with melanoma was significantly higher than that of the control. Among melanoma patients, the level of TIMP-1 with pT4 was significantly higher for patients who were: (i) pT1 and pT3; (ii) with LN metastasis (vs those without); (iii) in stage IV (vs those in stages I, II and III); and (iv) dead (vs those alive). The level of serum TIMP-2 in patients with melanoma was not different from the control. However, the level of TIMP-2 in patients with pT4 was significantly higher than for patients who were: (i) pT1, pT3 and control; (ii) with LN metastasis (vs those without metastasis and control); (iii) with stage IV (vs those in stages I and II and control); (iv) in recurrence (vs control); and (v) dead (vs those alive and control). These results suggest that increased serum levels of TIMP-1 and TIMP-2 reflected the extent of metastatic melanoma lesions, and that serum levels of TIMP-1 may be a new useful marker for melanoma progression.

Increased expression of germinal center-associated nuclear protein (GANP) is associated with malignant transformation of melanocytes.

BACKGROUND: Germinal center-associated nuclear protein (GANP) is a newly cloned molecule that is up-regulated in the germinal center B cells. Although GANP functions in the regulation of DNA repair during replication and survival of B cells, little is known about its expression in melanocytic cells. OBJECTIVES: To investigate whether GANP and phosphorylated-GANP (P-GANP) are expressed in cultured human melanocytes and melanoma cells and in benign and malignant melanocytic lesions. In addition, we aim to determine whether GANP and P-GANP are associated with malignant transformation of melanocytic lineage. METHODS: GANP and P-GANP expression in cultured melanocytic cells was analyzed by immunostaining and in vitro kinase assay. GANP and P-GANP expression in melanocytic lesions was analyzed by immunohistochemistry. RESULTS: GANP and P-GANP were up-regulated in cultured melanoma cells compared to melanocytes. GANP and P-GANP were restricted to nucleus of melanocytes but co-expressed in cytoplasm of melanoma cells. On the other hand, GANP and P-GANP were widely expressed at various levels in melanocytic nevi and melanoma lesions with nuclear and cytoplasmic staining pattern. Melanoma cells showed a stronger intensity of GANP and P-GANP than melanocytic nevus cells, however the staining intensity in primary melanoma lesions was not associated with any clinicopathological variables. Cytoplasmic GANP and P-GANP expression was associated with MCM3 and Ki67 expression. CONCLUSIONS: These data suggest, for the first time, that GANP and P-GANP are up-regulated in cultured melanoma cells compared to melanocytes and also they are widely expressed in benign and malignant melanocytic tumor cells.

Highly sensitive detection of melanoma at an early stage based on the increased serum secreted protein acidic and rich in cysteine and glypican-3 levels.

PURPOSE: There are no available tumor markers detecting primary melanoma at an early stage. The identification of such serum markers would be of significant benefit for an early diagnosis of melanoma. We recently identified glypican-3 (GPC3) as a novel tumor marker but could diagnose only 40% of melanomas. Thereby, we focused out attention on secreted protein acidic and rich in cysteine (SPARC) overexpressed in melanoma as another candidate for tumor marker. EXPERIMENTAL DESIGN: Secreted SPARC protein was quantified using ELISA in the sera from 109 melanoma patients, five patients with large congenital melanocytic nevus, 61 age-matched healthy donors, and 13 disease-free patients after undergoing a surgical removal. We also quantified GPC3 and 5-S-cysteinyldopa in the same serum samples and compared these markers for their diagnostic value. RESULTS: The serum SPARC concentrations in melanoma patients were greater than those in healthy donors (P = 0.001). When we fixed a cutoff value at the mean concentration plus 2 SD of the healthy donors, the serum SPARC was found to have increased in the sera of 36 of the 109 (33%) melanoma patients, whereas there were three (4.9%) false-positive cases of 61 healthy donors. Surprisingly, 19 of 36 patients showing increased SPARC levels were in stages 0 to II. The serum SPARC level decreased under the cutoff level in 10 of 13 patients after surgical removal. Using SPARC and GPC3 in combination thus enabled us to diagnose 47 of 75 (66.2%) melanoma patients at an early stage (0-II). CONCLUSIONS: SPARC or its combination with GPC3 is thus considered a potentially useful tumor marker, especially for melanoma at an early stage.

Giant malignant peripheral nerve sheath tumor of the scalp.

Herein, we describe a rare case of giant malignant peripheral nerve sheath tumor of the head in a 38-year-old Japanese man. The tumor measured 210 mm at its largest diameter and was ulcerated, hemorrhagic, multilocular and non-mobile. It should be noted that the patient stubbornly refused to see a doctor for a long time, resulting in the extreme growth of the tumor. We suspect a psychological basis for this behavior. Dermatohistopathological findings of the biopsy indicated ancient schwannoma and total excision was therefore performed. However, after 4 months, the patient developed multiple metastases and died. Post-mortem skin biopsy revealed features of malignant peripheral nerve sheath tumor. We performed immunohistochemical studies on the primary and recurrent lesions and concluded that there was a difference in the expression of Ki67 and p16. We propose that the expressions of Ki67 and p16 should be checked for all lesions of peripheral nerve sheath tumor for distinguishing benign from malignant forms.

A method to generate antigen-specific mAb capable of staining formalin-fixed, paraffin-embedded tissue sections.

Abnormalities in HLA class I antigen expression are frequently found in malignant tumors. Their potential role in the clinical course of the disease and in the outcome of T cell-based immunotherapy has stimulated interest in the characterization of the molecular mechanisms underlying HLA class I antigen abnormalities in malignant cells. Multiple mechanisms have been identified. Among them are abnormalities in antigen processing machinery (APM) component expression. In spite of this information, APM component expression in malignant lesions has been investigated only to a limited extent because of the lack of availability, for most APM components, of monoclonal antibodies (mAb) which stain formalin-fixed, paraffin-embedded tissues. The latter are the substrate of choice in immunohistochemical (IHC) reactions. To overcome this limitation, we have developed a simple and reproducible method to generate APM component-specific mAb which stain formalin-fixed, paraffin-embedded tissue sections. This method involves five steps: (i) immunogenic amino acid sequences, which display low homology with their mouse counterparts when possible, are identified in APM components and utilized to synthesize peptides; (ii) BALB/c mice are immunized with keyhole limpet hemocyanin (KLH)-conjugated synthetic peptides and with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)-purified recombinant APM component proteins; (iii) immunized mice, which develop high titer APM component-specific antibodies, are utilized to generate hybridomas which are screened for APM component-specific antibody production by Western blotting assays, with lymphoid cell lysates; (iv) identified APM component-specific mAb are characterized in their specificity and in their reactivity with permeabilized cells in ELISA and/or flow cytometry; and (v) mAb, with the appropriate reactivity pattern, are tested in IHC reactions with formalin-fixed, paraffin-embedded tissue sections. The use of the methodology we have developed resulted in the generation of a panel of APM component-specific mAb capable of staining formalin-fixed, paraffin-embedded tissue sections in IHC reactions. These reagents will facilitate the analysis of APM component expression in tissues under physiological and pathological conditions. In addition, the methodology we have described is likely to be applicable to other antigenic systems to develop mAb capable of detecting protein components of interest in formalin-fixed, paraffin-embedded tissue sections.

Human high molecular weight-melanoma-associated antigen (HMW-MAA): a melanoma cell surface chondroitin sulfate proteoglycan (MSCP) with biological and clinical significance.

The lack of effective conventional therapies for the treatment of advanced stage melanoma has stimulated interest in the application of novel strategies for the treatment of patients with malignant melanoma. Because of its expression in a large percentage of melanoma lesions and its restricted distribution in normal tissues, the high molecular weight-melanoma-associated antigen (HMW-MAA), also known as the melanoma chondroitin sulfate proteoglycan (MCSP), has been used to implement immunotherapy of melanoma. The potential clinical relevance of HMW-MAA/MCSP has stimulated investigations to characterize its structural properties and biological function in melanoma cells. Over the last 10 years, the field of HMW-MAA/MCSP research has seen tremendous growth. Specifically, a significant amount of information has been accumulated regarding (1) the structural characteristics of the HMW-MAA/MCSP, (2) its role in the biology of melanoma cells, and (3) the potential molecular mechanisms underlying the association between HMW-MAA/MCSP-specific immunity and survival prolongation in melanoma patients immunized with HMW-MAA/MCSP mimics. In this review, we summarize the characteristics of the HMW-MAA/MCSP in terms of its structure, antigenic profile, tissue distribution, and similarities with its counterparts in other animal species. Additionally, we discuss the role the HMW-MAA/MCSP plays in melanoma cell biology with emphasis on the recently identified signal transduction pathways triggered by the HMW-MAA/MCSP. Finally, we discuss the potential molecular mechanisms underlying the beneficial effect of anti-HMW-MAA/MCSP antibodies on the clinical course of the disease in patients with melanoma.

Clinical significance of MHC class II-associated invariant chain expression in human gastric carcinoma.

MHC class II antigens serve as restricted elements for cell presenting antigens to CD4+ helper T cells. CD4+ T cells and CD8+ cytotoxic T cells, which are tumor-infiltrating lymphocytes (TILs), and play a major role in the survey and attack against tumor cells in primary lesions. Invariant chain (Ii) has several functions in MHC class II-restricted antigen presentation. In addition, Ii is found to be closely involved in the regulation of anti-tumor immunity in several tumor types. However, the significance of Ii expression in tumor cells is not fully illustrated. Immunohistochemical staining of Ii expression was performed in 58 cases of human gastric carcinoma specimens. The prognostic analysis of patients with gastric carcinoma was also performed. A total of 67.2% (39/58) gastric carcinomas were found to be Ii-positive, whereas only 20.7% (12/58) showed positive immunoreactivity with anti-MHC class II determinants. Furthermore, Ii expression showed significant correlation with the differentiation of gastric carcinoma (p<0.05). Ii expression also showed an inverse correlation with the frequency of TILs around carcinoma tissues, as well as with the prognosis of gastric carcinoma (p<0.01). Ii expression is closely correlated with anti-tumor immunity in human gastric carcinoma. Therefore, Ii may serve as an independent clinical marker for poor biological behavior and prognostic analysis in patients with gastric carcinoma.

Identification of a novel human cancer/testis antigen, KM-HN-1, recognized by cellular and humoral immune responses.

PURPOSE: We used serologic screening of a cDNA expression library of human testis to identify novel cancer/testis antigens that elicit both humoral and cellular immune responses in cancer patients. EXPERIMENTAL DESIGN AND RESULTS: We identified a novel gene designated KM-HN-1 the expression of which is testis-specific among normal tissues; it contains coiled coil domains and a leucine zipper motif and encodes a putative protein consisting of 833 amino acids. KM-HN-1 expression was observed in various cancer tissues and cancer cell lines at both mRNA and protein levels. Immunofluorescence staining of an esophageal cancer cell line revealed that KM-HN-1 protein was present exclusively in the nucleus during mitosis. Recombinant KM-HN-1 protein was produced, and used for ELISA to quantitate levels of IgG antibody specific to KM-HN-1. Higher levels of IgG antibodies specific to KM-HN-1 were detected in many types and numbers of cancer patients but not in healthy donors. The CTL lines specific to KM-HN-1, generated from HLA-A*2402-positive healthy donors and cancer patients, killed human leukocyte antigen (HLA)-A24-positive cancer cells expressing KM-HN-1 but not cell lines that did not express either KM-HN-1 or HLA-A24. CONCLUSIONS: We identified a novel cancer/testis antigen, KM-HN-1, which elicited humoral immune responses in patients with various types of cancer. Furthermore, KM-HN-1-specific CTLs could be generated from both healthy donors and cancer patients, which indicated that KM-HN-1 can be a candidate for an ideal target for cancer immunotherapy.

Identification of glypican-3 as a novel tumor marker for melanoma.

PURPOSE: We reported recently the novel tumor marker glypican-3 (GPC3) for hepatocellular carcinoma. In the present study, we investigated the expression of GPC3 in human melanoma cell lines and tissues and asked whether GPC3 could be a novel tumor marker for melanoma. EXPERIMENTAL DESIGN: Expression of GPC3 mRNA and protein was investigated in human melanoma cell lines and tissues using reverse transcription-PCR and immunohistochemical analysis. Secreted GPC3 protein was quantified using ELISA in culture supernatants of melanoma cell lines and in sera from 91 patients with melanoma and 28 disease-free patients after surgical removal of primary melanoma. All of the subjects were Japanese nationals. RESULTS: In >80% of melanoma and melanocytic nevus, there was evident expression of GPC3 mRNA and protein. Furthermore, GPC3 protein was evidenced in sera of 39.6% (36 of 91) of melanoma patients but not in sera from subjects with large congenital melanocytic nevus (0 of 5) and from healthy donors (0 of 60). Twenty-seven of 36 serum GPC3-positive patients were negative for both serum 5-S-cysteinyldopa and melanoma-inhibitory activity, well-known tumor markers for melanoma. The positive rate of serum GPC3 (39.6%) was significantly higher than that of 5-S-cysteinyldopa (26.7%) and of melanoma-inhibitory activity (20.9%). Surprisingly, we detected serum GPC3 even in patients with stage 0 in situ melanoma. The positive rate of serum GPC3 at stage 0, I, and II (44.4%, 40.0%, and 47.6%) was significantly higher than that of 5-S-cysteinyldopa (0.0%, 8.0%, and 10.0%). Also observed was the disappearance of GPC3 protein in sera from 11 patients after surgical removal of the melanoma. CONCLUSIONS: GPC3 is apparently a novel tumor marker useful for the diagnosis of melanoma, especially in early stages of the disorder.

Protein kinase C alpha associates with phospholipase D1 and enhances basal phospholipase D activity in a protein phosphorylation-independent manner in human melanoma cells.

It is well known that phospholipase D plays a crucial part in the signal transduction of many types of cells, and is activated by protein kinase C alpha when cells are stimulated. To elucidate the role of phospholipase D in melanoma, the expression of phospholipase D1 and protein kinase C alpha in primary and metastatic lesions of acral lentiginous melanoma and superficial spreading melanoma was investigated using immunohistologic techniques. In addition, the mechanism of regulation of phospholipase D1 by protein kinase C alpha was examined in a human melanoma cell line HM3KO using an adenovirus-mediated gene transfer technique. Both phospholipase D1 and protein kinase C alpha were strongly expressed in primary and metastatic lesions of superficial spreading melanoma. Conversely, in acral lentiginous melanoma lesions, the expression of these two proteins increased dramatically with tumor progression; the expression of both phospholipase D1 and protein kinase C alpha was almost negative in the radial growth phase of primary acral lentiginous melanoma lesions, and increased synchronously in a progression-related manner in advanced acral lentiginous melanoma lesions, including vertical growth phase and metastatic lesions. Immunoprecipitation study showed that phospholipase D1 and protein kinase C alpha are associated physiologically in resting melanoma cells. Further immunoprecipitation study using HM3KO cells after adenovirus-mediated simultaneous overexpression of phospholipase D1 and protein kinase C alpha, or phospholipase D1 and the kinase-negative mutant of protein kinase C alpha revealed that both protein kinase C alpha and the kinase-negative mutant of protein kinase C alpha are associated with phospholipase D1 in melanoma cells in the absence of an external signal. Overexpression of protein kinase C alpha or the kinase-negative mutant of protein kinase C alpha in melanoma cells by the adenovirus vectors resulted in the enhancement of basal phospholipase D activity in a viral concentration-dependent manner. Furthermore, enhanced basal phospholipase D activity increased the in vitro invasive potential of HM3KO cells. These results suggest that upregulation of phospholipase D1 and protein kinase C alpha plays a part in the progression of acral lentiginous melanoma from the radial growth phase to the vertical growth phase. The present results also suggest that protein kinase C alpha associates with phospholipase D1 and enhances basal phospholipase D activity in a protein phosphorylation-independent manner in melanoma cells, which contributes to the cell's high invasive potential.

Two cases of unusual acral melanocytic tumors: illustration of molecular cytogenetics as a diagnostic tool.

The differential diagnosis between benign Spitz nevus and malignant melanoma may present considerable difficulties in some cases. Here we report 2 unusual melanocytic tumors with spitzoid features developing in acral sites of Japanese patients to illustrate the use of comparative genomic hybridization (CGH) to classify these lesions. Case 1 was a 12-mm-thick, >2 cm-diameter nodule on the sole of a 37-year-old man. Case 2 was a subungual tumor of the left index finger in a 13-year-old boy. CGH showed absence of chromosomal aberrations in case 1 and multiple aberrations in case 2, including focused amplification as previously described in acral melanomas. Case 1 was free of disease after 2.5 years of follow-up, whereas case 2 developed lymph node metastasis. We conclude that molecular techniques such as CGH can be of diagnostic help in the classification of histologically ambiguous lesions.

Repeated cationic multilamellar liposome-mediated gene transfer enhanced transduction efficiency against murine melanoma cell lines.

We investigated whether repeated cationic multilamellar liposome-mediated gene transfers enhanced the transduction efficiency against murine melanoma cell lines and experimental subcutaneous melanoma. In the former, the murine melanoma cell line, B16F10, was transfected by our original cationic multilamellar liposomes containing pVLacZ, which express beta-galactosidase in eukaryotic cells. Cells were exposed to the liposomes in a single, double, or triple procedure during the cell logarithmic proliferative period. We then evaluated the transduction efficiency by X-gal staining and beta-galactosidase assay. The number of positive cells and level of beta-galactosidase activity were significantly increased by repeated exposures compared with a single one. Cells transfected by the fluorescently labeled cationic liposome containing pEGFP-C1 showed both an increased uptake of liposomes and an increased number of EGFP expression cells following repeated exposures. In the latter, murine subcutaneous melanomas, which were made by transplantation of B16F10 in C57BL6 mice, were transfected by same liposomes. Subcutaneous melanomas were exposed to the liposomes in a single, double, or triple procedure. We then evaluated the transduction efficiency by the beta-galactosidase assay. The level of beta-galactosidase activity was significantly increased by repeated exposures compared with a single one. The results indicate that repeated exposures to the liposomes enhanced the transduction efficiency toward murine melanoma cells and experimental subcutaneous melanoma, and may provide a basis for the repeated-exposure protocol for human trials.

Comparison of phaeomelanin and its precursor 5-S-cysteinyldopa in the serum of melanoma patients.

5-S-Cysteinyldopa (5-S-CD) has been used as a biochemical marker of melanoma progression. Recently we have shown that the serum level of 5-S-CD is a sensitive and specific marker in predicting distant metastases. In melanocytes and melanoma cells, cysteinyldopa isomers are oxidized to phaeomelanin, the yellow to reddish melanin pigment. In this study we have developed a new method to measure levels of phaeomelanin in serum samples and have evaluated its clinical significance. The method is based on the production of 4-amino-3-hydroxyphenylalanine (4-AHP) and 3-amino-4-hydroxyphenylalanine (3-AHP) on reductive hydrolysis of phaeomelanin with hydriodic acid. 3-AHP is also derived from 3-nitrotyrosine-containing proteins. The isomeric 4-AHP and 3-AHP can be separated by high performance liquid chromatography. The mean +/- SD serum levels of 5-S-CD in control subjects (n = 36), in melanoma patients without recurrence (n = 92) and in melanoma patients with metastases (n = 24) were 2.7 +/- 1.2 nM (median 2.3 nM), 4.0 +/- 1.6 nM (median 3.8 nM) and 72 +/- 105 nM (median 35 nM), respectively. The serum levels of 4-AHP in these three groups were 45 +/- 21 nM (median 31 nM), 80 +/- 75 nM (median 53 nM) and 306 +/- 627 nM (median 133 nM), respectively. The serum levels of 4-AHP in patients with metastases (100 samples from 15 patients with progressive disease) correlated well (r = 0.887) with serum levels of 5-S-CD, while serum levels of 3-AHP did not (r = 0.240). The serum 5-S-CD and 4-AHP levels were serially analysed in the 15 patients with progressive disease. In two patients (13%), serum 4-AHP levels were elevated to abnormal levels before the serum 5-S-CD levels exceeded the cut-off value of 10 nM. In five patients (33%), the serum 4-AHP levels rose concurrently with the serum 5-S-CD levels. In the remaining eight patients (54%), serum 4-AHP levels were of less diagnostic value. Thus, the serum phaeomelanin level appears to be less sensitive than the serum 5-S-CD level in detecting distant metastases.

Growth inhibition of subcutaneous mouse melanoma and induction of natural killer cells by liposome-mediated interferon-beta gene therapy.

In this study we investigated the antitumour effect and mechanism of action of cationic liposome-mediated murine interferon-beta (IFNbeta) gene therapy in mouse B16F1 melanoma cells in vitro and in vivo. Murine IFNbeta gene transfer by cationic liposome resulted in substantial growth inhibition of B16F1 melanoma cells in culture when compared with phosphate buffered saline or recombinant murine IFNbeta treatment, or lacZ control gene transfer. Use of video-enhanced contrast-differential interference contrast (VEC-DIC) microscopy revealed that liposomes containing the murine IFNbeta gene [lip(pSV2muIFNbeta)], but not recombinant murine IFNbeta, induced dramatic morphological changes that characterize apoptosis, including bleb formation, shrinkage of cells, nuclear condensation and 'ballooning', in approximately 30% of the cells treated. Intratumoral administration of lip(pSV2muIFNbeta) resulted in a 5.5-fold reduction in the mean volume of subcutaneous melanoma lesions in syngeneic mice 15 days after treatment and eradicated the tumour in 18% of the mice treated. Immunocytochemical analysis demonstrated that a larger number of natural killer (NK) cells infiltrated the tumour following lip(pSV2muIFNbeta) treatment than in controls. In vivo depletion of NK cells using the anti-asialoGM1 antibody reduced the efficacy of lip(pSV2muIFNbeta) treatment. Taken together, our data suggest that cationic liposome-mediated IFNbeta gene therapy could be effective against melanoma by directly inducing cell death and stimulating NK cells.