Establishment of a C57BL/6 J mouse model of metastatic melanoma in the lung / 中国实验动物学报

Objective To investigate the influencing factors involved in the establishment of a C57BL/6 J model of metastatic melanoma in the lung,including the way of tumor inoculation,the number of inoculated cells and the time of tumor formation. Methods Mouse melanoma B16F10 cells were cultured in vitro. 1)Eighteen healthy male C57BL/6 J mice were randomly divided into three groups. Mice in each group received 100 μL cell suspension(including 3 ×106 melanoma cells)via intravenous,intraperitoneal and subcutaneous injection,respectively. After two weeks,the mice were killed and dissected,and the tumor growth and metastasis were observed. 2)Eighteen male mice were randomly divided into three groups. Mice in each group were injected with 3 ×106cells,1 ×106cells, and 3 ×105cells through the tail vein,respectively. After two weeks,mice were killed and dissected,and the tumor growth and metastasis were observed. 3)Eighteen male mice were randomly divided into three groups. Mice in each group were injected with 1×106cells though the tail vein. Mice were killed and dissected after one week, two weeks and three weeks, respectively. The growth and metastasis of tumor were observed. Results 1)The success rate of lung metastasis was 100% in the mice with intravenous injection,but not in mice receiving intraperitoneal injection and subcutaneous injection. 2)The size of metastatic melanoma nodules were moderate in mice inoculated by 1 ×106cells. The number of melanoma metastatic foci was too high in the mice inoculated with 3 ×106cells,but too low in the mice inoculated with 3 ×105cells. 3)Significant metastatic melanoma foci were observed in the mice killed and dissected after two weeks with no death. The number of melanoma foci in the lung was too high in the mice killed after three weeks,while was too low in the mice killed at one week after tumor cell inoculation. Conclusions Intravenous injection of 1×106mouse melanoma cells into C57BL/6 J mice and killed after two weeks is an optimal method for establishment of a mouse model of metastatic melanoma in the lung, and is worth of recommendation.

Research on 3-dimentional culture of human choroidal melanoma cell line (OCM-1) in vitro and the mechanism of vasculogenic mimicry / 眼科新进展

Objective To observe vasculogenic mimicry (VM) of human choroidal melanoma cell line OCM-1 cultured in vitro and the expression of PI3K and EphA2 protein,as well as to explore the possible mechanisms.Methods OCM-1 cells were cultured in vitro and stained with periodic acid Schiff (PAS) on days 7,which aimed to observe the formation of PAS-positive cyclic structures,that is,VM formation.Then immunohistochemical staining was performed to detect PI3K and EphA2 on day 1,4,7 and the results were observed.Ressults On day 4 of 3-demintional culture,most of OCM-1 cells were polygonal and the cytoplasm was abundant;the nuclei were round and the nucleoli were visible.A small part of the tumor cells were long spindle.It was found that several long spindle cells were connected to each other to form hemicyclic structure.After 7 days,a large number of tumor cells became long spindle,growing along the collagen scaffold,and long protrusions appeared,forming a ring structure.PAS staining showed that the tumor cells were mostly arranged in a row,and tumor cells imitated the formation of body blood vessels,resulting in cell band and pipeline-like cell layers,with one layer of extracellular matrix (PAS-positive substance) making up the ring structure.Moreover,the expression levels of PI3K and EphA2 on day 4 and 7 were significantly higher than those on day 1 (all P ＜ 0.05),and their expression levels on day 7 were higher than those on day 4 (all P ＜ 0.05).Conclusion EphA2 and PI3K may play an important role in the VM formation in 3-dimentional culture of human choroidal melanoma cell line OCM-1.

RNAi-mediated Human Nestin Silence Inhibits Proliferation and Migration of Malignant Melanoma Cells by G1/S Arrest via Akt-GSK3β-Rb Pathway / 华中科技大学学报（医学）（英德文版）

Human Nestin (hNestin) has been found to express in melanoma,and its expression is positively correlated with the advanced stage of melanoma.However,the precise role of hNestin in the development of melanoma has not been fully understood.The present study aimed to explore the role of hNestin in the proliferation and invasion of melanoma cells.The lentivirus vector carrying a short hairpin RNAs (shRNAs) targeting hNestin (hNestin-shRNA-LV) was stably infected into human melanoma cells UACC903,which expressed high levels of hNestin.The effects of hNestin knockdown on the proliferation,apoptosis,migration of melanoma cells and the related signaling pathways were investigated by immunofluorence,Western blotting and reverse transcription polymerase chain reaction (RT-PCR),respectively.The results showed that hNestin was expressed in most melanoma specimens and the melanoma cells studied.Knockdown of hNestin expression significantly inhibited the proliferation of melanoma cells,blocked the formation of cell colony,arrested cell cycle at G1/S stage and suppressed the activation of Akt and GSK3β.hNestin-silent cells also showed a sheet-like appearance with tight cell-cell adhesion,decreased membrane expression of N-cadherin and β-catenin,and attenuated migration.Furthermore,hNestin silence resulted in the inhibition of tumor growth in vivo.Our study indicates that hNestin knockdown suppresses the proliferation of melanoma cells,which might be through affecting Akt-GSK3β-Rb pathway-mediated G1/S arrest,and hNestin silence inhibits the migration by selectively modulating the expression of cell adhesion molecules in the process of epithelial-mesenchymal transition.

A preliminary study on the MATP gene knockout in a mouse melanoma cell line using CRISPR-Cas9 system / 中国比较医学杂志

Objective To knockout the MATP gene of mouse melanoma cell line B16F10 using CRISPR/Cas9 system,and to lay foundation for the functional study of MATP gene.Methods Specific primers of MATP were designed according to the report in http://crispr.mit.edu/ website.The primers were linked to pCAS9/gRNA1 vector.Then the positive vector was transfected into mouse melanoma B16F10 cells,and monoclonal cell lines were obtained by the infinite dilution method.After the genomes of different monoclonal cell lines were extracted and sequenced,the cell lines with MATP gene cleavage were screened,and the expression of MATP in these cell lines was verified by Western-blot analysis.Results Three MATP gene knockout cell lines were successfully obtained.The western-blot results showed that the cell lines did not express MATP protein.Conclusions The knockout of MATP gene in B16F10 cell line can be successfully achieved using the pCAS9/gRNA1 vector.

Cytotoxic effects of local anesthesia through lidocaine/ropivacaine on human melanoma cell lines / Efeitos citotóxicos de anestesia local com lidocaína/ropivacaína em linhagens celulares de melanoma humano

Abstract Background: Local anesthetics (LAs) are generally considered as safe, but cytotoxicity has been reported for several local anesthetics used in humans, which is not well investigated. In the present study, the cytotoxicity of lidocaine, ropivacaine and the combination of lidocaine and ropivacaine were evaluated on human melanoma cell lines. Melphalan, a nitrogen mustard alkylating agent, was used as a control agent for comparison of cytotoxic activity. Methods: Melanoma cell lines, A375 and Hs294T, were exposed to 1 h to different concentrations of above agents. Cell-viability after exposure was determined by flow cytometry. Results: Investigated LAs showed detrimental cytotoxicity on studied melanoma cell lines in time- (p < 0.001), concentration- (p < 0.001), and agent dependant. In both A375 and Hs294T cell lines, minimum cell viability rates were found after 72 h of exposure to these agents. Lidocaine 2% caused a reduction of vital cells to 10% ± 2% and 14% ± 2% in A375 and Hs294T, respectively after 72 h of exposure. Ropivacaine 0.75% after 72 h reduced viable cells to 15% ± 3% and 25% ± 3% in A375 and Hs294T, respectively. Minimum cell viability after 72 h exposure to the combination was 10% ± 2% and 18% ± 2% in A375 and Hs294T, respectively. Minimum cell viability after 72 h exposure to melphalan was 8% ± 1% and 12% ± 2%, in A375 and Hs294T, respectively. Conclusion: LAs have cytotoxic activity on human melanoma cell lines in a time-, concentration- and agent-dependant manner. Apoptosis in the cell lines was mediated through activity of caspases-3 and caspases-8.

Resumo Justificativa: Os anestésicos locais (ALs) são geralmente considerados como seguros, mas citotoxicidade foi relatada em vários anestésicos locais usados em seres humanos, a qual não é bem investigada. No presente estudo, a citotoxicidade de lidocaína e ropivacaína e da combinação de lidocaína e ropivacaína foi avaliada em linhagens celulares de melanoma humano. Melfalano, um agente alquilante de mostarda nitrogenada, foi usado como um agente de controle para a comparação da atividade citotóxica. Métodos: Linhagens celulares de melanoma, A375 e Hs294T foram expostas por uma hora a concentrações diferentes dos agentes mencionados acima. A viabilidade celular após a exposição foi determinada por citometria de fluxo. Resultados: Os ALs investigados mostraram citotoxicidade prejudicial nas linhagens celulares de melanoma estudadas dependente do tempo (p < 0,001), da concentração (p < 0,001) e do agente. Em ambas as linhagens de células A375 e Hs294T, níveis mínimos de viabilidade celular foram encontrados após 72 horas de exposição a esses agentes. Lidocaína a 2% provocou uma redução das células vitais para 10% ± 2% e 14% ± 2% em A375 e Hs294T, respectivamente, após 72 horas de exposição. Ropivacaína a 0,75% após 72 horas reduziu as células viáveis para 15% ± 3% e 25% ± 3%, em A375 e Hs294T, respectivamente. A viabilidade celular mínima após exposição de 72 horas para a combinação foi de 10% ± 2% e 18% ± 2% em A375 e Hs294T, respectivamente. A viabilidade celular mínima após exposição de 72 horas ao melfalano foi de 8% ± 1% e 12 ± 2, em A375 e Hs294T, respectivamente. Conclusão: Os ALs têm atividade citotóxica em linhagens de celulares de melanoma humano de modo dependente do tempo, da concentração e do agente. A apoptose nas linhagens celulares foi mediada por meio da atividade das caspases-3 e caspases-8.

Effect of silencing HlF-1αby siRNA on the expression of erythropoietin in malignant melanoma of the choroid cell under hypoxia / 国际眼科杂志(Guoji Yanke Zazhi)

AlM: To silent hypoxia inducible factor-1α ( HlF-1α) gene in malignant melanoma of the choroid cell by small interference RNA ( siRNA ) and investigate its effect on the expression of matrix metalloproteinase-2 ( MMP-2 ) in the choroid cell line human uveal melanoma cell (OCM-1) in hypoxia environment.METHODS:OCM-1 cells cultured on culture flask were divided into normal group and hypoxia group. Hypoxia group were divided into five groups: simple hypoxic group, and interference group, and negative control group, and positive control group, and liposome group. Normal group cells were cultured on DMEM culture flask with 10% FBS, 100U/mL penicillin, 100μg/mL streptomycin as well as high concentration of glucose. The cells were maintained at 37℃ in a humidified 5% CO2 incubator. Cells in good condition were selected for experiment. For hypoxia group, chemical hypoxia inducer CoCl2 was added into nutrient medium at the concentration of 100μmol/L to simulate hypoxia microenvironment. We designed and synthesised siRNA ( siRNA + negative control+positive control ) , the target sequences of the HlF-1α to transfect hypoxic malignant melanoma of the choroid cell. SiRNA including HlF-1α siRNA, β-actin siRNA and negative control group synthesized in vitro transfected hypoxic OCM - 1 cell through Lipofectamine2000. The expression of HlF-1α, MMP-2 gene and the protein were detected by RT-PCR and Western blot. RESULTS: Compared with the normal group, the expression of HlF-1α mRNA was not obviously changed (P>0. 05), but the expression of HlF-1α protein and MMP- 2 mRNA protein was significantly higher ( P0. 05).CONCLUSlON: Hypoxia status may upregulate the HlF-1α in OCM-1 cells by increasing the expression of protein. Hypoxia can also inactivate MMP-2, resulting in upregulation of MMP-2 RNA and the expression of its protein. The expression of HlF-1α and MMP-2 mRNA can be down-upregulated by transfecting OCM-1 with HlF-1α siRNA.

OAZI-1 overexpression in mouse B16-F1 melanoma cells can effectively stimulate DC immune activity / 中国免疫学杂志

Objective:To analyze, at cellular level, whether the mouse B16-F1 melanoma cells with OAZI-1 overexpression could activate antigen-presenting cells and promote the phagocytotic and antigen-presenting efficiencies of mouse peritoneal macrophage and bone marrow derived DC on tumor cells. Methods:The plasmid pcDNA3. 1(+)/OAZI-1 was transfected into B16-F1 cells by Li-pofectamine2000 reagent. The positive clones with OAZI-1 overexpression ( B16/OAZI-1 ) were identified by Western blot assay and RT-PCR. Macrophages from abdominal cavity and DC from bone marrow were collected from BALB/c mouse. The B16-F1 cells transfected with the pcDNA3. 1(+) (B16/3. 1) were used as the control cells in this experiment. B16-F1 cells and macrophages were co-cultured for 4 h at a 1∶5 ratio and DC were co-cultured with B16-F1 cells at 1∶1 ratio for 4 h. And then the phagocytotic efficiencies were assayed by flow cytometry. DC were co-cultured with B16-F1 cells at 1∶1 ratio for 24 h and then the expression of mature DC surface marker molecules CD40,CD80,CD86 were determined by flow cytometry. The DC activated by the tumor cells were co-cultured with mouse spleen lymphocytes for 24 h, and then IFN-γ content in culture medium was analyzed by ELISA. Results: Phagocytotic assay showed that,compared to the control cells,the OAZI-1 overexpression in B16-F1 cells significantly enhanced the engulfment of B16-F1 cells by macrophages ( 24. 7% vs 53. 9% ) and DC ( 8. 2% vs 13. 8%) . When DC were co-incubated with OAZI-1 overexpressed B16-F1 for 24 h,the expression levels of CD40,CD80,CD86 on the DC surface,which were the molecular markers for matured DC,increased from 24. 2%,20. 8% and 16. 4% to 46. 8%,32. 5% and 36. 1% respectively. Co-culture of tumor-activated DC with the spleen lymphocytes resulted in an increased IFN-γcontent in the culture medium(32. 9 pg/ml vs 15. 1 pg/ml). Conclusion:The tumor cells with OAZI-1 overexpression can be engulfed more efficiently by macrophages and DC. And this process can induce the maturation and activation of DCs. Matured DC could induce T cell activation and then activate the anti-tumor immune response.

Effects of all-trans retinoic acid, acitretin and tazarotene on apoptosis and active caspase-3 in human melanoma cells A375 / 中华医学美学美容杂志

Objective To investigate the effects of all -trans retinoic acid (ATRA),acitretin and tazarotene on apoptosis and active caspase-3 of human melanoma cells A375 and to discuss the relevant significance.Methods The effects of retinoids on apoptosis and active caspase-3 of A375 cells were examined in vitro.Early apoptosis analysis by double staining with annexin V-FITC and PI,and active caspase 3 analysis by the staining of FITC conjugated monoclonal rabbit anti active caspase-3 antibody were measured by flow cytometer.Results ATRA and acitretin induced apoptosis of A375 cells (both P＜0.05),but tazarotene was not effective (P＞0.05).ATRA and acitretin elevated the cell population with detectable active caspase-3 (both P＜0.05),but tazarotene was not effective either (P＞0.05).Acitretin played a most prominent role among the retinoids. Early apoptosis ratio was significantly and positively correlated with active caspase-3 percentage in both ATRA and acitretin (P＜0.05).Conclusions Acitretin and ATRA induce apoptosis of A375 cells in which caspase is critical.Retinoids are prospectively applied as an assisting alternative for treating melanoma.

Preferential Cytotoxic Effect of Genistein on G361 Melanoma Cells Via Inhibition of the Expression of Focal Adhesion Kinase

Resistance to the induction of apoptosis is a possible mechanism by which tumor cells can survive anti-neoplastic treatments. Melanoma is notoriously resistant to anti-neoplastic therapy. Previous studies have demonstrated focal adhesion kinase (FAK) overexpression in melanoma cell lines. Given its probable role in mediating resistance to apoptosis, many researchers have sought to determine whether the downregulation of FAK in melanoma cells would confer a greater sensitivity to anti-neoplastic agents. Genistein is a known inhibitor of protein-tyrosine kinase (PTK), which may attenuate the growth of cancer cells by inhibiting the PTK-mediated signaling pathway. This present study was undertaken to investigate the effect of genistein on the expression of FAK and cell cycle related proteins in the G361 melanoma cell line. Genistein was found to have a preferential cytotoxic effect on G361 melanoma cells over HaCaT normal keratinocytes. Genistein decreased the expression of 125 kDa phosphotyrosine kinase and the FAK protein in particular. Genistein treatment did not affect the expression of p53 in G361 cells in which p21 is upregulated. The expression of cyclin B and cdc2 was downregulated by genistein treatment. Taken together, our data indicate that genistein induces the decreased proliferation of G361 melanoma cells via the inhibition of FAK expression and regulation of cell cycle genes. This suggests that the use of genistein may be a viable approach to future melanoma treatments.

S Phase Cell Cycle Arrest and Apoptosis is Induced by Eugenol in G361 Human Melanoma Cells

Eugenol is an essential oil found in cloves and cinnamon that is used widely in perfumes. However, the significant anesthetic and sedative effects of this compound have led to its use also in dental procedures. Recently, it was reported that eugenol induces apoptosis in several cancer cell types but the mechanism underlying this effect has remained unknown. In our current study, we examined whether the cytotoxic effects of eugenol upon human melanoma G361 cells are associated with cell cycle arrest and apoptosis using a range of methods including an XTT assay, Hoechst staining, immunocytochemistry, western blotting and flow cytometry. Eugenol treatment was found to decrease the viability of the G361 cells in both a time- and dose-dependent manner. The induction of apoptosis in eugenol-treated G361 cells was confirmed by the appearance of nuclear condensation, the release of both cytochrome c and AIF into the cytosol, the cleavage of PARP and DFF45, and the downregulation of procaspase-3 and -9. With regard to cell cycle arrest, a time-dependent decrease in cyclin A, cyclin D3, cyclin E, cdk2, cdk4, and cdc2 expression was observed in the cells after eugenol treatment. Flow cytometry using a FACScan further demonstrated that eugenol induces a cell cycle arrest at S phase. Our results thus suggest that the inhibition of G361 cell proliferation by eugenol is the result of an apoptotic response and an S phase arrest that is linked to the decreased expression of key cell cycle-related molecules.

Apoptotic Effects of A Cisplatin and Eugenol Co-treatment of G361 Human Melanoma Cells

Eugenol (4-allyl-2-methoxyphenol) is a naturally occurring phenolic compound that is widely used in dentistry as a component of zinc oxide eugenol cement that is commonly applied to the mouth environment. Cisplatin is one of the most potent known anticancer agents and shows significant clinical activity against a variety of solid tumors. This study was undertaken to investigate the synergistic apoptotic effects of co-treatments with eugenol and cisplatin on human melanoma (G361) cells. To investigate whether this co-treatment efficiently reduces the viability of G361 cells compared with each single treatment, an MTT assay was conducted. The induction and augmentation of apoptosis were confirmed by DNA electrophoresis, Hoechst staining and an analysis of DNA hypoploidy. Western blot analysis and immunofluorescent staining were also performed to evaluate the expression levels and the translocation of apoptosis-related proteins following this co-treatment. Furthermore, proteasome activity and mitochondrial membrane potential (MMP) changes were also assayed. The results indicated that a co-treatment with eugenol and cisplatin induced multiple pathways and processes associated with an apoptotic response in G361 cells including nuclear condensation, DNA fragmentation, a reduction in MMP and proteasome activity, the increase and decrease of Bax and Bcl-2, a decreased DNA content, the release of cytochrome c into the cytosol, the translocation of AIF and DFF40 (CAD) into the nucleus, and the activation of caspase-9, caspase-7, caspase-3, PARP and DFF45 (ICAD). In contrast, separate treatments of 300 microM eugenol or 3 microM cisplatin for 24 h did not induce apoptosis. Our present data thus suggest that a combination therapy of eugenol and cisplatin is a potential treatment strategy for human melanoma.

The establishment of the model in ICR mice with B16 melanoma of C57BL/6 mice / 医学研究生学报

Objectives: To establish melanoma models of B16 cell line in ICR mice. Methods: Inoculating B16 Melanoma cells in ICR mice's feet, back, abdomen and caudal vein, to establish transplanted and metastatic melanoma models. We observed the morphologic characteristics and examined the pathological parameters of melanoma. We analyzed the relations between the number of B16 cells inoculated and the genesis of tumors, the influence of tumor growth on mice's behavior,and growth curve of the melanoma. Results:Six days later, sesamoid, linear and nodal even kaulifloweroid melanoma were seen gradually in ICR mice's feet, back, abdomen and lung (anatomid) after inoculating. The bearing tumor rates have a positive relation to the number of inoculated B16 cells. The rates of bearing tumor was near 100%, after inoculating with 5?10 6 B16 cells. Conclusions: The B16 melanoma cell line derived from C57/B16 mice(black ) has high rates of bearing tumor in ICR mice (white). The B16 melanoma models in ICR mice are ideal tumor models that have the characteristics of easy established,easy observing and easy obtaining.

Effect of QS Alex 755 nm Laser on the Expression of p16INK4a on the Melanoma Cell Lines / 中华皮肤科杂志

Objective To investigate the effect of QS Alex 755 nm laser on the p16INK4a expression of melanoma cell lines. Methods Cultured melanoma cell lines, HTB 66, Sk-mel-24, G361 were irradiated with different doses of QS Alex 755 nm laser (0.85 ~ 2 J/cm2) and the irradiated cells were harvested 24 h after irradiation. The expression of p16INK4a protein and mRNA levels were detected by flowcytometer and RT-PCR respectively. Results Compared with the nonirradiated cells the level of p16INK4a protein in HTB 66 cell line increased significantly after laser irradiation, and no change in p16INK4a-negative cell line (Sk-mel-24) and less laser-sensitive cell line (G361). Loss of p16 functional activity leading to disruption of the cell cycle arrest was observed in cell line HTB 66. RT-PCR analysis indicated that p16INK4a mRNA increased in a dose-dependent manner in HTB 66. The p16INK4a mRNA expression was not changed in p16INK4a protein negative Sk-mel-24 and G361 cell lines. Conclusions QS Alex 755 nm laser increases the expression of p16INK4a protein in HTB 66 cell line, which may imply that survival cells after laser therapy may undergo DNA damage. Hence, the laser therapy should not be recommended to treat congenital nevi among those with high risk of aberrant p16.

Molecular analysis of HLA-DR gene expression induced by IFN-gamma in malignant melanoma cell lines

Human leukocyte antigen (HLA) class II molecules are polymorphic cell surface glycoproteins that are crucial for the cellular interaction in immune response. The expression of class II molecules is regulated in a tissue-specific and cytokine-inducible manner, and is mainly restricted to the antigen presenting cells. However, some tumor cells also express class II molecules, and in some class-II-negative tumor cells, class II expression is inducible by interferon (IFN)-gamma. However, their expression varies, even though the tumor cells originate from the same histological origin; some tumor cells show strong expression, others show weak or no expression. To determine whether this differential expression of class II molecules on tumor cells is transcriptionally regulated, FACS analysis and Northern hybridization were performed using a panel of melanoma cell lines, IGR3, Malme-3M, SK-Mel-24, and SK-Mel-28 to analyze the cell surface expression and mRNA transcription rate of HLA-DR before and after treatment with IFN-gamma. FACS analysis showed that before IFN-gamma treatment, IGR3 and Malme-3M cells barely expressed HLA-DR. On the contrary, almost all of the SK-Mel-24 cells (> 90%) and a relatively high rate (> 50%) of SK-Mel-28 cells expressed HLA-DR. After IFN-gamma treatment, HLA-DR expression was induced in Malme-3M cells and SK-Mel-28 cells which displayed elevated levels of HLA-DR expression in a time-dependent manner. However, IGR3 cells never responded to IFN-gamma. Northern analysis showed that treatment with IFN-gamma led to the steady-state mRNA augmentation of the HLA-DR gene in Malme-3M and SK-Mel-28, whereas in IGR3, IFN-gamma did not augment the transcriptional rate of the HLA-DR gene. To further clarify this differential modulation, sequencing analysis of PCR product of the HLA-DR proximal promoter region was done, since the transcription rate of the class II gene is controlled by the well-conserved proximal promoter region. Six independent clones from PCR products of the HLA-DRA proximal promoter region and 16 clones from PCR products of the HLA-DRB proximal promoter region were isolated from the above cell lines and sequenced. Comparison of the nucleotide sequences of all 6 clones of DRA promoter showed that the sequences are extremely similar in both regulatory sequences and their intervening sequences. Sixteen clones of HLA-DRB promoter showed sequence variations such as substitution and insertion/deletion, and these 16 clones could be further grouped into 6 homologues with sequence homology. These data established that the melanoma cell lines studied here showed a differential susceptibility to IFN-gamma on the modulation of HLA-DR molecules, that this modulation is transcriptionally regulated, and that the difference in promoter activity by sequence variation might contribute to such a differential transcriptional regulation at the promoter level.

Expression of Heat Shock Protein 70 Family in Melanocytes / 대한피부과학회지

BACKGROUND: When cells or organisms are exposed to environmental stresses, they respond by synthesizing a characteristic group of proteins called heat shock proteins(HSP) or stress proteins. In a variety of HSP, the so-called HSP 70 family is the most prominent, conserved, and best characterized. The HSP 70 family is required for survival of cells during and after thermal stress. OBJECTIVE: The purpose of this study is to investigate if the cultured human melanocytes and rnelanotic malignant melanoma cell lines(SK 30) expressed HSP 70 family unstressed, after heat shock and ultraviolet exposure. METHODS: Protein was isolated from melanocytes and SK 30. Western blotting was done for identification of the HSP 70 family. RESULTS: HSP 70 family expression could be detected in the unstressed cultured human melanocytes and SK 30(malignant melanoma cell lines). HSP 70 family expression inereased in the melanocytes and SK 30 after heat shock. Irradiation of the melanocytes with UVA resulted in a decrease in expression of HSP 70 family after 32, 48 J/cm compared with 4, l6 J/cm. Irradiation of the melanocytes with UVA + B resulted in a dose-dependent increase in expression of HSP 70 family but a decrease in expression of HSP 70 family after 80mJ/cm. Irradiation of SK 30 with UVA resulted in a dose-dependent decrease in expression of the HSP 70 family. CONCLUSION: HSP 70 family expression was detected even unstressed. This high base line HSP 70 family expression may suggest that melanocytes have ability to protect from environmental stresses like keratinocytes.

Expression of Heat Shock Protein 70 Family in Melanocytes / 대한피부과학회지

BACKGROUND: When cells or organisms are exposed to environmental stresses, they respond by synthesizing a characteristic group of proteins called heat shock proteins(HSP) or stress proteins. In a variety of HSP, the so-called HSP 70 family is the most prominent, conserved, and best characterized. The HSP 70 family is required for survival of cells during and after thermal stress. OBJECTIVE: The purpose of this study is to investigate if the cultured human melanocytes and rnelanotic malignant melanoma cell lines(SK 30) expressed HSP 70 family unstressed, after heat shock and ultraviolet exposure. METHODS: Protein was isolated from melanocytes and SK 30. Western blotting was done for identification of the HSP 70 family. RESULTS: HSP 70 family expression could be detected in the unstressed cultured human melanocytes and SK 30(malignant melanoma cell lines). HSP 70 family expression inereased in the melanocytes and SK 30 after heat shock. Irradiation of the melanocytes with UVA resulted in a decrease in expression of HSP 70 family after 32, 48 J/cm compared with 4, l6 J/cm. Irradiation of the melanocytes with UVA + B resulted in a dose-dependent increase in expression of HSP 70 family but a decrease in expression of HSP 70 family after 80mJ/cm. Irradiation of SK 30 with UVA resulted in a dose-dependent decrease in expression of the HSP 70 family. CONCLUSION: HSP 70 family expression was detected even unstressed. This high base line HSP 70 family expression may suggest that melanocytes have ability to protect from environmental stresses like keratinocytes.

Induction of ICAM-1 and HLA-DR expression by IFN-gamma in malignant melanoma cell lines

Two human malignant melanoma cell lines, Malme-3M and SK-Mel-28, were analyzed for their ability to induce the expression of intercellular adhesion molecule 1 (ICAM-1) and human leukocyte antigen (HLA)-DR molecules on their cell surfaces as well as at the transcriptional level before and after treatment with interferon (IFN)-gamma. Both cell lines demonstrated a high percentage(> 99%) of ICAM-1 expression regardless of IFN-gamma treatment. Before IFN-gamma treatment, Malme-3M cells barely expressed HLA-DR molecules ( 50%) of HLA-DR expression. Both cell lines displayed elevated levels of HLA-DR expression in a time dependent manner after IFN-gamma treatment. However, these two cell lines have been shown to respond differentially to IFN-gamma. The molecular mechanism underlying such a differential behavior was investigated, and HLA-DR gene regulation was studied at the transcriptional level. Treatment with IFN-gamma led to the steady-state mRNA augmentation of the HLR-DR gene. The HLA-DRA mRNA augmentation was similar in both cell lines, whereas in Malme-3M, IFN-gamma did not augment the rate of transcription of the HLA-DRB gene as much as in SK-Mel-28. Data from this study established the fact that the melanoma cell lines displayed a differential susceptibility to IFN-gamma on the modulation of HLA-DR molecules, and this modulation was transcriptionally regulated.

Effect of cytokines on the expression of cell adhesion molecule and on the adhesion of melanoma cells to endothelial cells

We examined the role of cell adhesion molecules (CAM) by which tumor cells bind to the endothelial cells using human umbilical vein endothelial cells (HUVEC) and cultured melanoma cells. Endothelial cells from human umbilical veins were isolated and examined for CAM expression and its modulation by tumor necrosis factor-alpha (TNF-alpha), interleukin-1 (IL-1), interleukin-6 (IL-6) or interferon-gamma (IFN-gamma). The expression of intercellular adhesion molecule 1 (ICAM-1) on HUVEC was increased by TNF-alpha, IL-1 and IFN-gamma when measured by ELISA or flow cytometric (FACS) analysis. IL-6 did not increase ICAM-1 expression on HUVEC. Two melanoma cell lines, Malme-3M and SK-Mel-28, showed increased expression of ICAM-1 after treatment with TNF-alpha, IL-1 and IFN-gamma in FACS analysis. IFN-gamma induced increased expression of HLA-DR only in SK-Mel-28 melanoma cells, not in Malme-3M melanoma cells. Neither HUVEC nor melanoma cells expressed lymphocyte function-associated antigen 1 (LFA-1) in either the basal (i.e., cytokine untreated) condition or the cytokine treated condition. Melanoma cells showed minimal increment in adhesion to TNF-alpha or IL-1 treated HUVEC than to cytokine untreated HUVEC. HUVEC and melanoma cells did not express LFA-1 and increased ICAM-1 expression by TNF-alpha, IL-1 and IFN-gamma treatment in FACS analysis did not coincide with minimal increase of melanoma cells adhesion to cytokine treated HUVEC. These results suggest that adhesion between melanoma cells and HUVEC is probably mediated by molecular interaction other than ICAM-1/LFA-1.

In vitro inhibition of trichosanthin-monoclonal antibody conjugate on human melanoma cells / 中国免疫学杂志

We have constructed an immunotoxin(Ng76-TCS),which was composed of a monoclonalantibody directed against human melanoma and trichosanthin(TCS)——a single chain ribosomeinactivating protein.The cultured human melanoma cells(M21)were inhibited effectively byNg 76-TCS.The cytotoxicity of Ng76-TCS to M21 cells was 2,000-fold higher than that of free TCS and Ng76 mixture.A conjugate,which was prepared with normal mice immunoglobulinand TCS(NIgG-TCS),was 160-fold less cytotoxic to M21 cells.Meanwhile Ng76-TCS was125-fold less cytotoxic to nontarget cells Hela.These results showed that the immunotoxinNg76-TCS was a potent and specific anti-human melanoma agent.

Proliferation of Mel-CAM defined IT in first-trimester villi and hydatidiform mole / 西安交通大学学报(医学版)

Objective To explore the relationship between Mel-CAM and adherence and invasion of trophoblastic cells and proliferation of Mel-CAM defined intermediate trophoblast.Methods Double immunohistochemical staining technique was used to detect the expression of Ki-67 in Mel-CAM defined intermediate trophoblast in human villi during the first trimester and hydatidiform mole.Results In this study there was a significantly stronger expression of Mel-CAM in hydatidiform mole than that in first-trimester villi (P