Melanocytes express 3G5 surface antigen

The 3G5-reactive ganglioside antigen (3G5 antigen) is expressed on the surface of various cell types including pericytes, pancreatic islet cells, thyroid follicular cells, and cells of the pituitary and the adrenal medulla. Expression on melanocytes has not yet been reported. We examined 148 5-microm cryosections of 12 normal skin samples and 45 skin tumors (21 melanocytic nevi, 8 malignant melanoma primaries, 4 metastases of malignant melanoma, 3 basal cell carcinomas, and 9 pigmented seborrheic keratoses) by triple fluorescence technique with the monoclonal antibody 3G5, DNA fluorochrome, and the anti-melanocytic antibody A103 (Anti-Melan-A). In normal skin, 3G5 reactivity was detected in epidermal melanocytes of 4 of 12 cases with 14.8 +/- 24.1% positive melanocytes; 20 of 21 nevi (72.2 +/- 29.1% positive nevus cells, mean +/- SD), 8 of 8 primary melanomas (83.9 +/- 12.3% positive melanoma cells), and 4 of 4 melanoma metastases (82.5 +/- 6.5% positive melanoma cells) expressed the 3G5 antigen. All tumor cells of investigated basal cell carcinoma or seborrheic keratosis were 3G5 negative. This is the first report of 3G5 antigen expression in melanocytes. The data demonstrate high expression of this ganglioside in the aggregated melanocytes of malignant or benign tumors but low or absent expression in singular melanocytes (normal epidermis, seborrheic keratoses) reflecting a different biologic state.     

Modulation of intercellular adhesion molecule-1 expression on human melanocytes and melanoma cells: evidence for a regulatory role of IL-6, IL-7, TNF beta, and UVB light.

Intercellular adhesion molecule-1 (ICAM-1) is involved in cell-cell interactions of leukocytes and parenchymal cells and thus plays an important role in immunologic and inflammatory reactions. The expression of ICAM-1 that is found on many different cells such as melanocytes and melanoma cells is induced by various cytokines, including interferon-gamma (IFN gamma), interleukin (IL)-1 and tumor necrosis factor alpha (TNF alpha). Because expression of ICAM-1 in melanoma was found to correlate with increased risk of metastasis, the regulation of ICAM-1 expression on human melanocytes and melanoma cells was investigated. Foreskin-derived melanocytes and melanoma cell lines (A375, G361) were incubated with different cytokines and ICAM-1 expression was evaluated by fluorescence-activated cell sorter. IFN gamma, IL-1, IL-7, TNF alpha, and TNF beta significantly upregulated ICAM-1 expression in a dose-dependent manner. Most interestingly, the cytokine IL-6, which does not influence adhesion-molecule expression on other cells, significantly upregulated melanocyte and melanoma cell ICAM-1 expression. This effect was dose dependent and could be blocked by an IL-6 antibody. Irradiation with ultraviolet (UVB) light did not influence constitutive ICAM-1 expression on melanoma cells and melanocytes, but suppressed cytokine-induced ICAM-1 expression when cells were harvested 16 h after irradiation. These findings were further confirmed by Northern blot analysis, showing a marked accumulation of ICAM-1 mRNA after cytokine treatment, which was reduced by irradiation with UVB light. However, when UVB-exposed melanoma cells were cultured for at least 48 h induction of ICAM-1 expression was observed. These data indicate that, similar to other cells, ICAM-1 expression on melanoma cells and melanocytes is regulated by cytokines and that UVB light affects ICAM-1 expression on melanocytic cells in a biphasic manner.     

Heterogeneity of cytokine production by human malignant melanoma cells

Recent investigations indicate that malignant melanoma cells can produce distinct cytokines. While differences in the production of single cytokines have been observed among different melanoma cell lines, the extent of variability in the production of single and multiple cytokines between individual melanoma cell lines has not been as thoroughly investigated. A heterogeneity in melanoma cell cytokine production could have important implications for the biology of this aggressive neoplasm since certain cytokines may act as autocrine growth factors or be potent modulators of host immune response to the developing tumor. The purpose of this study is to assess the cytokine production profile of two widely available human melanoma cell lines, A375 and G361. The A375 cell line constitutively expressed the mRNA for IL-1 alpha, IL-1 beta and PDGF-A, with increased expression of these cytokines after induction with PMA. GM-CSF mRNA was expressed by the A375 melanoma line only after induction with PMA. No IL-6 mRNA was detected in the A375 melanoma cell line. The cell culture supernatants from the A375 cells likewise contained a parallel increase in IL-1 activity as determined in the D10 bioassay and secreted GM-CSF and PDGF-AA as measured by ELISA. In contrast, the G361 cell line did not express IL-1, GM-CSF or PDGF-A mRNA (constitutively or after PMA induction) but expressed only IL-6 mRNA and secreted IL-6 activity after PMA induction. These results demonstrate a significant heterogeneity in the production of IL-1 alpha, IL-1 beta, IL-6, GM-CSF, and PDGF in two distinct melanoma cell lines. This study demonstrates that individual melanoma cell lines express and secrete multiple cytokines both constitutively and after stimulation with PMA. The immunodulating and mitogenic properties of these melanoma-derived cytokines may have implications in determining the biologic behavior of different malignant melanomas.     

Tumor-infiltrating CD3- NK cells are more effective than CD3+ T cells in killing autologous melanoma cells.

We have studied the phenotype and functional activity of tumor-infiltrating lymphocytes (TIL) derived from eight human melanomas cultured for up to 60 d in the presence of recombinant IL-2. In the early period of the cultures, TIL were predominantly T cells of CD8+ phenotype and contained 10-30% of CD3- cells. Four of the five early TIL cultures tested in a cytotoxicity assay displayed a degree of MHC-unrestricted lysis on a series of autologous and allogenic melanoma cell lines as well as the K562 natural killer-sensitive target. With longer periods of time in culture, all TIL lines showed a decrease in lytic activity that was associated with the loss of CD3- cells. Thus, most of the killing of short-term TIL cultures appeared to be mediated by CD3- natural killer cells, whereas CD3+ T cells were found to be weak anti-tumor effectors. Even though the CD3+ T cells were not cytotoxic on K562 targets, their lytic activity (even weak) against melanoma cells appeared to be non-MHC restricted, and was blocked by anti-CD3 antibodies. In addition, cytotoxicity of the CD3+ TIL cultures was compared to that of a CD3-/NKH1+ cell line purified from peripheral blood. It was found that natural killer cells were much more potent than CD3+ TIL on the melanoma cell lines tested.     

NK cell density in malignant skin tumours—a stereological study

We determined the density of natural killer (NK) cells in frozen sections of malignant melanoma (nine cases), squamous cell carcinoma (six cases) and non-neoplastic tonsil (five cases) by immunohistological and stereological methods. The mean density of NK cells in malignant melanoma (5.0 +/- 1.5 X 10(3)/mm3) and in squamous cell carcinoma (5.1 +/- 2.7 X 10(3)/mm3) was significantly lower than in germinal centres of normal human tonsils (5.0 +/- 0.8 X 10(4)/mm3). The NK cell/tumour cell ratio (0.03 +/- 0.015 in malignant melanoma and 0.02 +/- 0.015 in squamous cell carcinoma) is 10(2)-10(4) times lower than that commonly used for in vitro assays. The possible role of NK cells in malignant skin tumours should be viewed with caution.     

Inducible nitric oxide synthase is expressed in normal human melanocytes but not in melanoma cells in response to tumor necrosis factor-alpha,interferon-gamma,and lipopolysaccharide

Nitric oxide is a gaseous messenger involved in the regulation of several physiologic processes in various cell types, including skin cells. Three different nitric oxide synthases (neuronal nitric oxide synthase, endothelial nitric oxide synthase, inducible nitric oxide synthase) have been identified in human cells. For inducible nitric oxide synthase, an inducibility by cytokines and lipopolysaccharides have been found. For murine melanoma cells, a connection between elevated levels of nitric oxide after inducible nitric oxide synthase induction and consequent apoptosis had been described. By northern analysis, we detected inducible nitric oxide synthase mRNA in four of 15 human melanoma cell lines cultured without inducible nitric oxide synthase inducing cytokines. Induction of inducible nitric oxide synthase mRNA by tumor necrosis factor-alpha, interferon-gamma, and lipopolysaccharides was seen in normal human melanocytes but not in melanoma cell lines. In accordance, inducible nitric oxide synthase protein expression was clearly inducible in cultures of normal melanocytes, whereas in six melanoma cell lines investigated, inducible nitric oxide synthase was found weakly expressed already before treatment with tumor necrosis factor-alpha, interferon-gamma, and lipopolysaccharides, and its expression was not inducible. The apoptotic rates both in normal melanocytes and in two melanoma cell lines (SK-Mel-19 and O-Mel-2) were increased by treatment with tumor necrosis factor-alpha, interferon-gamma, and lipopolysaccharides; however, these effects could not be prevented by the specific nitric oxide synthase inhibitor N(G)-monomethyl-L-arginine. These data reveal a clear-cut difference between human melanoma cell lines and cultured normal human melanocytes with respect to inducible nitric oxide synthase inducibility. Although the data do not support the hypothesis that inducible nitric oxide synthase is an important regulator for apoptosis in human melanoma cells, the regulation deficiency found for melanoma cells may be of importance for melanocytic transformation and tumor progression.     

抗原处理相关转运体和MHC-Ⅰ类分子在黑素瘤细胞株的表达

目的 检测抗原处理相关转运体（TAP）及主要组织相容性复合体（MHC）-Ⅰ类分子在恶性黑素瘤细胞系中的表达。方法 采用Western印迹法、RT-PCR和流式细胞仪分别检测TAP蛋白、mRNA和MHC-Ⅰ类分子在3株恶性黑素瘤细胞系（A375、A875、KZ28）中的表达,并与正常黑素细胞做对照。结果 与正常黑素细胞相比,TAP mRNA表达在A375、A875、KZ28细胞系中均显著降低,t值分别为5.89,4.45和4.57,P值均 < 0.01;TAP 蛋白的表达均明显降低,t值分别为5.46,4.32和4.67,P值均 < 0.01。在A375细胞中TAP mRNA和蛋白下降最显著。MHC-Ⅰ类分子也具有相同的趋势,t值分别为6.16,5.22和5.61,P值均 < 0.01。结论 TAP蛋白及其mRNA在A375、A875、KZ28细胞系中表达显著降低,可能为恶性黑素瘤细胞逃逸机体免疫监视的一条途径。     

Changes in Immunological Parameters after Combination Adjuvant Therapy with Intravenous DTIC,ACNU, and VCR,and Local Injection of IFN-β (DAV + IFN-β Therapy) into Malignant Melanoma

Combination adjuvant therapy with intravenous dimethyl triazeno imidazole carboxamide (DTIC), 1-[4-amino-2-methyl-5-pyrimidinyl]-methyl-3-[2-chloroethyl]-3-nitrosourea hydrochloride (ACNU) and vincristine (VCR) and local injection of interferon-β (IFN-β) (DAV + IFN-β therapy) has been widely applied to treat malignant melanoma, and its therapeutic effect is accepted in Japan. Natural killer (NK) activity, CD4+ and CD8+ T cell counts, CD4/CD8 ratio, and white blood cell counts were analyzed before and after DAV + IFN-β therapy in order to validate its efficacy. After DAV + IFN-β therapy, the CD4/CD8 ratio was elevated; however, numbers of both CD4+ and CD8+ T cells and NK activity were consecutively depressed. Peripheral lymphocytes were also decreased, possibly by myelosuppression due to the DAV therapy. The posttreatment suppression of NK activity appeared in spite of the administration of IFN-β. It is suggested that a more effective adjuvant immunomodulator should be introduced to improve the therapeutic effect of the combination adjuvant chemotherapy in malignant melanoma.     

Protein kinase C-betaII represses hepatocyte growth factor-induced invasion by preventing the association of adapter protein Gab1 and phosphatidylinositol 3-kinase in melanoma cells

The hepatocyte growth factor (HGF) signaling pathway was examined in human normal melanocytes and three malignant melanoma cell lines. HGF-induced activation of c-Met, its receptor-tyrosine kinase, was observed in both melanocytes and melanoma cells, whereas phosphatidylinositol 3-kinase (PI3K), a downstream target of c-Met, was not activated in the melanocytes but enhanced in the melanoma cell lines. The electrophoretic mobility of Gab1, the scaffolding adapter protein that couples activated c-Met and PI3K, was slower in the melanocytes than that in the melanoma cells, and the mobility shifted to that of the melanoma cells after treatment with alkaline phosphatase, indicating that Gab1 is highly phosphorylated on serine and threonine in the melanocytes. Introduction of protein kinase C (PKC)-betaII into the melanoma cells, which is expressed in melanocytes but absent in melanoma cells, resulted in serine and threonine phosphorylation of Gab1 and also prevented tyrosine phosphorylation of Gab1 and its association with PI3K. Furthermore, the introduction of PKC-betaII suppressed HGF-induced activation of PI3K, and attenuated the in vitro invasion activity of the melanoma cells. These results indicate that the HGF signaling process from Gab1 to PI3K is negatively regulated by PKC-betaII, and its loss is critical for melanoma cells to gain invasive potential.     

Gangliosides of melanoma.

The relationships between the ganglioside composition of melanomas and their biologic behavior were investigated. (1) The amount of GM2 and/or GD2 in melanoma cells injected into nude mice correlated with the tumor growth rate. (2) GD2 content of melanoma cell lines correlated with sensitivity to radiation and vincristine. (3) GM2 expression of melanoma cells correlated with sensitivity to lymphokine-activated killer cells. (4) Gangliosides inhibited the proliferation of human T cells stimulated with interleukin-4 or interleukin-2. Based on these results, we proposed a hypothesis for the role of melanoma-associated gangliosides in the biologic behavior of melanomas and suggested a prospective melanoma treatment related to the gangliosides.     

In vitro Comparison between Mouse B16 and Human Melanoma Cell Lines of the Expression of ICAM-1 Induced by Cytokines and/or Hyperthermia

The expression of intercellular adhesion molecule-1 (ICAM-1) in mouse B16 and human melanoma cell lines was investigated by indirect immunofluorescence using a FACScan analyzer. Mouse B16 melanoma cell lines (B16-F1 and F10) did not express ICAM-1 under ordinary culture conditions. Neither in vitro hyperthermia at 41°C for 3 or 6 hr nor cytokines such as γ interferon (IFN-γ) or tumor necrosis factor α (TNF-α) induced ICAM-1 expression in B16 melanoma cell lines. A combination of IFN-γ with TNF-α caused slight induction of ICAM-1 expression in the B16-F10 melanoma cell line. Hyperthermia at 41°C for 3 hr in combination with the cytokines induced a slight expression of ICAM-1 in the B16-F1 melanoma cell line. Hyperthermia at 41°C for 3 hr or 6 hr did not induce de novo ICAM-1 expression but hyperthermia at 43°C for 6 hr caused rather suppression of the expression of ICAM-1 in the three human melanoma cell lines tested. In contrast, they showed a clear increase in the expression of ICAM-1 after treatment with either with IFN-γ or TNF-α, and the expression was further augmented by a combination of the two cytokines. Treatment with cytokines in combination with hyperthermia at 41°C or 43°C for 3 hr did not augment the expression of ICAM-1 over that in cytokine-treated human melanoma cell lines, at normal temperatures. Thus, it is concluded that mouse B16-F1 and F10 melanoma cell lines are different from human melanoma cell lines in terms of induction of ICAM-1 expression by cytokines and/or hyperthermia.     

CXCR7在常见皮肤恶性肿瘤及其细胞株中的表达及意义

目的 探讨趋化因子受体CXCR7在皮肤鳞状细胞癌、基底细胞癌、侵袭性皮肤黑素瘤及其细胞株中的表达及其意义。方法 收集30例皮肤鳞状细胞癌、25例基底细胞癌、30例皮肤黑素瘤的癌组织,采用免疫组织化学方法,检测CXCR7蛋白表达水平。采用RT-PCR、细胞免疫组化方法检测CXCR7在A375、M14、A431、HaCaT细胞株中mRNA及蛋白水平。结果 CXCR7在侵袭性皮肤黑素瘤中表达明显,高表达率为80%（24/30）,皮肤鳞状细胞癌及基底细胞癌分别为26.67%（8/30）、8%（2/25）;皮肤黑素瘤CXCR7高表达率与鳞状细胞癌、基底细胞癌比较,差异均有统计学意义（χ2值分别为17.16和28.36,P值均 < 0.05）。CXCR7 mRNA在A375、M14、A431细胞株中均可检出,其中A375表达最强,而HaCaT细胞不表达;细胞免疫组化显示,仅在A375细胞见棕黄色颗粒着色。结论 皮肤黑素瘤及其细胞株A375高表达CXCR7,其可能参与了其恶性侵袭与转移。     

MV3细胞系体外构建恶性黑素瘤模型的研究

目的 探讨MV3黑素瘤细胞接种至人去表皮的真皮（DED）上,体外构建皮肤恶性黑素瘤模型中黑素瘤细胞的侵袭方式。方法 制备含部分基底膜成分的人去表皮的真皮组织（de?鄄epidermized dermise, DED）,利用PAS染色和Ⅳ型胶原免疫组化染色鉴定基底膜。将MV3黑素瘤细胞制成细胞瘤液,接种于DED表面,采用液下培养和空气－液面培养相结合的方式进行培养,用HE及免疫组化染色（S-100蛋白和HMB45）,观察黑素瘤细胞在DED的分布。结果 肉眼观察,DED在接种MV3黑素瘤细胞后颜色无明显变化。经PAS染色和Ⅳ型胶原免疫组化染色,验证DED中含有基底膜成分,分别位于DED表面和其内的皮肤附属器管壁上。组织学及免疫组化染色检查显示：MV3黑素瘤细胞在DED表面的基底膜带上生长分布,并在凹陷处形成大小不等团、灶;在皮肤附属器管壁内,呈环形、带状,贴附于基底膜上;在DED侧面,黑素瘤细胞弥散性浸润生长。组织中黑素瘤细胞S-100蛋白阳性和HMB45弱阳性。结论 通过皮肤器官培养方式,可构建皮肤恶性黑素瘤的体外模型,基底膜影响恶性黑素瘤的侵袭生长方式。     

Identification of genes specifically regulated in human melanoma cells

In order to improve the characterization of human malignant melanoma cells and their variant gene expression in vitro, a search for specifically regulated genes was performed. Four melanoma cell lines (M5, MEWO, IGR39, SKMEL13) and newly cultured normal human melanocytes were included in a comparative hybridization (differential screening) of a human melanoma cDNA-library. Six cDNAs were isolated showing a stronger expression (four genes) or a weaker expression (two genes) in melanoma cells than in normal human melanocytes. Quantification of the expression patterns of the two repressed genes in Northern blots revealed general expression in all melanocyte cultures examined, no expression in three cell lines (M5, IGR39, SKMEL13) and weak expression in MEWO. The four induced genes were found to be only weakly expressed in normal human melanocytes, but showed an elevated expression in all of the four melanoma cell lines tested. Thus, using the technique of differential screening, consistent gene regulation at the messenger RNA level was identified, which distinguishes the four melanoma cell lines tested from normal melanocytes. We conclude from the expression patterns that specific gene regulation in melanoma cells in vitro is characterized both by strong repression of some melanocyte genes, as well as by the induction of other genes, but there was no indication of new expression of genes specific for melanoma cells. Because of the uniform induction or repression in different melanoma cell lines, it is conceivable that the cloned genes may be involved in the malignant transformation of melanocytic cells.     

MV3恶性黑素瘤皮肤体外模型中基质金属蛋白酶-2、9的表达

目的 探讨体外构建的MV3恶性黑素瘤皮肤模型中基质金属蛋白酶-2（MMP-2）和基质金属蛋白酶-9（MMP-9）的表达及意义。方法 用MV3恶性黑素瘤细胞接种至去表皮的真皮（DED）上,采用液下和空气－液面培养相结合的方式进行组织培养,2周后取培养的黑素瘤组织切片分别做HE染色及MMP-2、9免疫组化染色。同时用RT-PCR对培养组织进行MMP-2、9表达检测。结果 HE染色显示MV3黑素瘤细胞在DED表面呈条带状生长,在凹陷处聚集生长,在DED残留的皮肤附属器管腔中形成大小不等的团、灶;免疫组化染色显示在MV3黑素瘤细胞生长的DED表层及DED附属器管壁基底膜处MMP-2、9均呈阳性表达;RT-PCR检测进一步证实体外构建的恶性黑素瘤组织表达MMP-2和MMP-9,而未接种MV3黑素瘤细胞的DED组织无MMP-2、9表达。结论 体外构建的恶性黑素瘤皮肤模型中瘤细胞侵入到真皮,表达MMP-2、9,为进一步研究黑素瘤的侵袭与转移提供实验依据。     

Expression of GD3 disialoganglioside antigen on peripheral T-lymphocytes in patients with disseminated malignant melanoma

Abstract Disialoganglioside antigens GD2 and GD3 are expressed on most melanoma cells. On melanoma surrounding T-cells in immunohostological sections, disialogangliosides can also be found, as well as in a small % of T-lymphocytes in peripheral blood from healthy persons. In order to find out if there is a difference in ganglioside expression on peripheral T-lymphocytes between melanoma patients and healthy persons, we examined the expression of CD3 as T-lymphocytic antigen and GD2 or GD3 antigens, respectively, by flow cytometry. We used peripheral mononuclear blood cells of 12 patients with advanced disseminated malignant melanoma and of 12 healthy control donors. For immunostaining, murine monoclonal antibodies Leu-4, 14G2a and MB3.6 were used, recognizing CD3, GD2 and GD3. GD2 expression was found on only a low proportion of T-lymphocytes in patients and healthy persons (pat.: mean = 1.2%± 0.7%, co.: mean = 0.4%± 0.4%). Disialoganglioside antigen GD3, however, could be demonstrated on an average of 8.4%± 4.6% of patients' and on 4.0%± 2.1% of healthy persons' T-cells. There is a statistically significant difference (P < 0.01) between the data of patients' and control group. We conclude that there is a correlation between advanced malignant melanoma and expression of GD3 antigen on patients' peripheral T-lymphocytes. The immunological relevance of our findings is discussed.