The terminal differentiation of malignant melanoma cells is known to be induced by activating cAMP signaling pathway with alpha-MSH or cAMP analogues. However, sustained activation of cAMP signaling system that induces the differentiation of melanoma cells, also induces the desensitization of the pathway at the receptor level. Nevertheless, the adaptation of adenylate cyclase (AC) expression by sustained activation of cAMP signaling system has not been clearly understood. This study was performed to examine whether the sustained activation of cAMP system induce changes in the expression AC isoforms as an adaptation mechanism. Treatment of B16/F10 murine melanoma cells with 100 mM forskolin for 6 days resulted in differentiation, melanin accumulation and increased expression of tyrosine hydroxylase mRNA. In the forskolin-treated melanoma cells, change in expression of various AC isoform at the transcription level was detected by reverse-transcription polymerase chain reaction (RT-PCR). Expression of AC isoform mRNA: ACI, III, VI, VII, and IX increased to the level of 196-392% of the control whereas the level of ACII was decreased by 30%. The cAMP concentration was increased both in basal and alpha-MSH stimulated cells, but the AC activity was decreased in the forskolin treated cells. Thus, these results suggest that sustained activation of cAMP system induces differential expression of AC isoforms, which results in increase of cAMP accumulation.
Adenylate Cyclase/*genetics ; Animal ; Cell Differentiation ; Cyclic AMP/*metabolism ; Forskolin/*pharmacology ; Isoenzymes/genetics ; Melanoma, Experimental/*enzymology/*pathology ; Mice ; Signal Transduction

Though the malignancy of a tumor is generally postulated to be affected by the degree of differentiation of tumor cells, the relationship between differentiation and malignancy of tumors has not been clearly elucidated. Using in vitro established mouse(B16) and human(IGR3) melanoma cell lines, we performed various in vitro and in vivo experiments to clarify the relationship between melanogenicity and malignancy. High and low melanin-producing clones were selected from both cell lines by the limiting dilution technique and their melanogenicities were confirmed by the determination of melanin quantity and tyrosinase activity along with electron microscopic examination. Selected clones from both cell lines revealed that low melanin-producing clones showed a slightly broader chromosomal distribution, a shorter doubling time with a higher DNA synthesis and a greater colony forming capacity in semi-solid agar medium than those of high melanin-producing clones. The low melanin-producing clone derived from B16 also had a lower tumor-take dose and a more rapid tumor-growth rate than the high melanin-producing counterpart following transplantation into syngeneic mice. These results support the concept that the melanogenicity and other biological characteristics associated with malignancy are inversely related in malignant melanoma cells.
Animal ; Cell Line ; Human ; Melanins/*metabolism ; Melanoma/*pathology ; Melanoma, Experimental/*pathology ; Mice ; Mice, Inbred Strains ; Microscopy, Electron ; Neoplasm Transplantation ; Skin Neoplasms/*pathology ; Tumor Cells, Cultured/*pathology ; Tumor Stem Cell Assay

AIMTo investigate the possibility of using stealth liposomes modified with arginine-glycine-aspartic acid (RGD) mimetic as the targeted carriers to achieve increased accumulation in tumor and enhanced intracellular delivery for the encapsulated anticancer drugs.

METHODSRGD mimetic (RGDm) as a ligand for integrins was synthesized and covalently conjugated to the active PEGylated phospholipids (DSPE-PEG-BTC) to form RGDm conjugate (DSPE-PEG-RGDm). Then RGDm-modified SL (RGDm-SL) containing DOX (RGDm-SL-DOX) and SL containing DOX (SL-DOX) were prepared by film dispersion followed by ammonium sulfate gradient method. The pH-sensitive probe, BCECF-AM, was used to study the binding of melanoma cells to DSPE-PEG-RGDm. Flow cytometry and confocal microscopy were performed to evaluate the cellular association or DOX uptake for RGDm-SL-DOX or SL-DOX in vitro.

RESULTSThe melanoma cells A375 and B16 showed enhanced binding to the immobilized DSPE-PEG-RGDm. The cells treated with RGDm-SL-DOX showed remarkable increase in cellular association or DOX uptake compared with SL-DOX.

CONCLUSIONThe RGDm-modified SL could be as the targeted carriers to facilitate the delivery of the encapsulated anti-cancer drugs into tumor cells by receptor-mediated way.

Animals ; Antibiotics, Antineoplastic ; administration & dosage ; metabolism ; Cell Adhesion ; Cell Line, Tumor ; Cell Nucleus ; metabolism ; Doxorubicin ; administration & dosage ; metabolism ; Drug Carriers ; Drug Delivery Systems ; Humans ; Liposomes ; Melanoma, Experimental ; metabolism ; pathology ; Oligopeptides ; Phosphatidylethanolamines ; Polyethylene Glycols

OBJECTIVETo establish a syngeneic mouse model of liver tumor stably expressing hepatitis B virus (HBV) antigens.

METHODSMelanoma cell line B16 cells were transfected with pLXSN-2HBV. Cells (named B16/HBV) stably and persistently expressing HBV surface (HBsAg) and core (HBcAg) antigens were identified. The cells were injected into the hepatic subcapsular space of fifteen C57BL/6J mice. The mice were divided into 3 groups, receiving 100, 1000 or 5000 cells in a total volume of 5 µl per mouse, respectively, five mice in each group. Two weeks after the tumor cell inoculation, serum samples from the mice were collected weekly and the serum concentration of HBsAg and anti-HBs was quantified by ELISA. The tumor growth in the mouse liver was monitored by a high-resolution ultrasound system. Expression of HBsAg and HBcAg in the tumor tissues was determined by immunohistochemistry.

RESULTSLiver tumors were formed in all the mice receiving 1000 and 5000 B16/HBV cells per mouse, and in 80% of the mice receiving 100 B16/HBV cells. HBsAg and anti-HBs were detectable in their sera from 2 weeks after tumor cell inoculation. The mice receiving 100 cells per mouse began to die 4 weeks, those receiving 1000 cells per mouse began to die 3 - 4 weeks and those receiving 5000 cells began to die 2 - 3 weeks after the cell inoculation. All the tumor cells expressed HBsAg and HBcAg.

CONCLUSIONSThe B16/HBV cells stably and persistently express HBV antigens both in vitro and in vivo. A mouse model of transplanted liver tumor stably expressing HBV antigens has been successfully established by inoculation of those cells into the hepatic subcapsular space.

Animals ; Cell Line, Tumor ; Disease Models, Animal ; Female ; Hepatitis B Core Antigens ; metabolism ; Hepatitis B Surface Antigens ; metabolism ; Hepatitis B e Antigens ; metabolism ; Hepatitis B virus ; genetics ; metabolism ; Liver Neoplasms, Experimental ; immunology ; virology ; Melanoma, Experimental ; metabolism ; pathology ; Mice ; Mice, Inbred C57BL ; Neoplasm Transplantation ; Plasmids ; Recombinant Proteins ; genetics ; metabolism ; Transfection

OBJECTIVETo investigate the inhibitory effect of DNA vaccine immunization on neu-overexpressed melanoma growth in prophylactic treatment and anti-lung-metastasis experiments in C57BL/6 mice.

METHODSpcDNA-neu transfected into B16F10 with transfection reagent Fugene 6, neu-overexpressed cell clone B16F10-neu was selected with limited dilution method. The growth curve was drawn to analyse its proliferating character in vitro. With Helios gene gun system, DNA vaccine pWRG-neu was immunized to 8-week-old C57BL/6 mice in the shaved abdominal skin for 3 times at two-weekly interval. After immunization, the life span was analyzed. Using MTT assay, the cytolysis activity of the DNA immunized mice spleen cells was compared.

RESULTSOne clone of neu-overexpressed B16F10-neu was selected and its proliferating character was the same as B16F10 and B16F10-pcDNA. In prophylactic, treatment and anti-lung-metastasis experiments, gene gun-mediated pWRG-neu immunization could exhibit antitumor effects. The growth and metastasis of neu-overexpressed melanoma was reduced dramatically. The spleen cells of the immuned mice showed cytotoxic T lymphocyte (CTL) activity.

CONCLUSIONGene gun-mediated gene transfer is effective and practicable. DNA vaccine pWRG-neu is potent in preventing subsequent tumor cells challenge, inhibiting the tumor growth and metastasis.

Animals ; Biolistics ; Cell Line, Tumor ; Cytotoxicity, Immunologic ; Genes, erbB-2 ; Immunization ; Lung Neoplasms ; prevention & control ; secondary ; Melanoma, Experimental ; metabolism ; pathology ; therapy ; Mice ; Mice, Inbred C57BL ; Neoplasm Transplantation ; Plasmids ; Receptor, ErbB-2 ; metabolism ; T-Lymphocytes, Cytotoxic ; immunology ; Vaccines, DNA

We constructed a recombinant adenoviral vector expressing human tissue inhibitors of metalloproteinase-1(TIMP-1), and evaluated the inhibition of TIMP-1 secreted by primary fibroblasts after infection with adenovirus-mediated TIMP-1 gene (Ad-TIMP-1) on tumor cell invasion and metastasis in mouse melanoma. It was found that TIMP-1 was detected in the supernatants of cultured mouse primary fibroblasts after infection with Ad-TIMP-1 by indirect enzyme-linked immunosorbent assay (ELISA). The TIMP-1 secreted by Ad-TIMP-1 infected primary fibroblast significantly inhibited B16BL6 cell invasion and metastasis both in vitro and in vivo. We also demonstrated that the primary fibroblasts transfected by Ad-TIMP-1, after being subcutaneously injected into mouse, can secreted TIMP-1 into the blood of mouse and maintained at the therapeutic in vivo levels of TIMP-1. These results suggest that the preparation of Ad-TIMP-1 infected primary fibroblast be an effective method to deliver TIMP-1 gene in vivo, which provides a new strategy of gene therapy and has the potential for clinical applications in the treatment of tumor cell metastasis.
Adenoviridae ; genetics ; metabolism ; Animals ; Female ; Fibroblasts ; metabolism ; Genetic Therapy ; Humans ; Melanoma, Experimental ; pathology ; therapy ; Mice ; Mice, Inbred C57BL ; Neoplasm Invasiveness ; Neoplasm Metastasis ; Recombinant Proteins ; biosynthesis ; genetics ; pharmacology ; Tissue Inhibitor of Metalloproteinase-1 ; biosynthesis ; genetics ; pharmacology

In order to enhance the antitumor efficacy of recombinant Newcastle disease virus, rNDV-IL15 was rescued in this study. Recombinant plasmid prNDV-IL15 was constructed, and BHK21 cells were transfected with the recombinant plasmid. Finally, the recombinant Newcastle disease virus rNDV-IL15 was successfully rescued. The growth curves of these two recombinant viruses were determined. Murine melanoma B16F10 cells were infected with rNDV-IL15 at MOI of 0.1, and the expression level of IL15 in the supernatant was detected by ELISA. The antitumor efficacy of rNDV-IL15 and rNDV was compared in vitro and in vivo. Results showed that prNDV-IL15 was constructed and recombinant virus rNDV-IL15 was successfully rescued. The growth curve of rNDV-IL15 showed that the growth of rNDV-IL15 had not been changed after insertion of IL15 gene. Results showed that there was high level of IL15 expression in the supernatant of rNDV-IL5-infected B16F10 cells (1 044.3 +/- 27.7 ng x mL(-1)). rNDV-IL15 and rNDV significantly inhibited the growth of B16F10 cells in vitro in a time-dependent manner. However, there was no significant difference between them. In animal experiments, rNDV-IL15 efficiently suppressed tumor growth in vivo when compared with rNDV, and the difference was statistically significant. The results suggested that rNDV-IL15 is a more effective antitumor agent.
Animals ; Body Weight ; Cell Line, Tumor ; Cell Proliferation ; Chick Embryo ; Cytotoxicity, Immunologic ; Female ; Genetic Therapy ; Interleukin-15 ; genetics ; metabolism ; Melanoma, Experimental ; pathology ; therapy ; Mice ; Neoplasm Transplantation ; Newcastle disease virus ; genetics ; Plasmids ; Recombinant Proteins ; genetics ; metabolism ; Transfection ; Tumor Burden

This study was aimed to construct the CD14 eukaryotic expression vector, establish the transgeneic CD14 positive cell line in order to facilitate the establishment of a mouse model of antibody targeting therapy for human acute monocytic leukemia (AML-M(5)). Total RNA extracted from peripheral blood mononuclear cells was treated with RNAase-free DNAase, the human CD14 gene was cloned and sequenced through the RT-PCR and T-A clone techniques. Eukaryotic expressional vector pcDNA3.1(+)/CD14 was constructed by cleaving with double restriction endonuleases and ligating with T4 ligase. A murine melanoma cell line B16 was transfected with the pcDNA3.1(+)/CD14 recombinant with Superfect transfection reagent. Positive clones were selected by G418 and the expression of human CD14 on the transfectant was confirmed by flow cytometry (FCM). The results indicated that the sequence of the human CD14 cDNA cloned was exact to be same as the one from GenBank database. The recombinant pcDNA3.1(+)/CD14 was identified with double-enzyme cleaving. The expression of the human CD14 on the transfectant (B16/CD14) was confirmed by FCM. In conclusion, the murine cell line B16/CD14 fransfected with human CD14 gene has been established which can be used for the study of human AML-M(5) antibody targeting therapy with mouse model.
Animals ; Antigens, Neoplasm ; biosynthesis ; genetics ; Eukaryotic Cells ; metabolism ; Gene Transfer Techniques ; Genetic Vectors ; genetics ; Humans ; Leukemia, Monocytic, Acute ; genetics ; Lipopolysaccharide Receptors ; genetics ; Melanoma, Experimental ; genetics ; metabolism ; pathology ; Mice ; Mice, Inbred C57BL ; Transfection ; Tumor Cells, Cultured

The increasing incidence and mortality associated with advanced stages of melanoma are cause for concern. Few treatment options are available for advanced melanoma and the 5-year survival rate is less than 15%. Targeted therapies may revolutionize melanoma treatment by providing less toxic and more effective strategies. However, maximizing effectiveness requires further understanding of the molecular alterations that drive tumor formation, progression, and maintenance, as well as elucidating the mechanisms of resistance. Several different genetic alterations identified in human melanoma have been recapitulated in mice. This review outlines recent progress made in the development of mouse models of melanoma and summarizes what these findings reveal about the human disease. We begin with a discussion of traditional models and conclude with the recently developed RCAS/TVA somatic cell gene delivery mouse model of melanoma.
9,10-Dimethyl-1,2-benzanthracene ; Animals ; Avian Leukosis Virus ; genetics ; Avian Proteins ; genetics ; metabolism ; Cell Line, Tumor ; Disease Models, Animal ; Gene Transfer Techniques ; Genetic Vectors ; genetics ; Humans ; Melanocytes ; metabolism ; Melanoma ; genetics ; pathology ; Melanoma, Experimental ; chemically induced ; genetics ; Mice ; Mice, Transgenic ; Neoplasm Transplantation ; Receptors, Virus ; genetics ; metabolism ; Skin Neoplasms ; genetics ; pathology ; Tetradecanoylphorbol Acetate ; Transgenes

OBJECTIVETo investigate the distribution of regulatory T-lymphocytes in the splenocytes cocultured with syngeneic low-immunogenic tumor cells, as compared with that of highly-immunogenic tumor cells, to investigate the mechanism underlining tumor evasion.

METHODSThree different immunogenic tumor cells were cocultured with syngeneic splenocytes individually to mimic cancer immunity in vitro. The proliferation response of splenocytes was measured by thymidine incorporation. The distribution of TR cells, CD4(+) IFN-gamma (+) T cells and CD4(+) IL-10(+) T cells were analyzed by flow cytometry. The secretion of IFN-gamma and IL-10 in supernatants was measured by ELISA assay.

RESULTSThe stimulation Index of splenocytes cocultured with syngeneic highly-immunogenic H22 or FBL3 was much higher than that of poorly immunogenic melanoma D5. In each group, stimulation Index of splenocytes cocultured with allogeneic tumor cells was higher than that of the corresponding tumor immunity model. In addition, compared with those of highly-immunogenic tumors, there were more TR, CD4(+)IL-10(+) and less CD4 (+)IFN-gamma(+) T cells in the splenocytes, and higher IL-10 and lower IFN-gamma levels in the supernatant of the splenocytes stimulated with low-immunogenic D5 cells.

CONCLUSIONPoorly-immunogenic tumor cells can induce the proliferation of TR cells, which may play an important role in tumor evasion.

Animals ; CD4 Antigens ; immunology ; Cell Line, Tumor ; Cell Proliferation ; Coculture Techniques ; Female ; Interferon-gamma ; metabolism ; Interleukin-10 ; metabolism ; Interleukin-2 Receptor alpha Subunit ; immunology ; Leukemia, Experimental ; pathology ; Liver Neoplasms ; pathology ; Melanoma, Experimental ; pathology ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Spleen ; cytology ; T-Lymphocytes, Regulatory ; cytology ; immunology