As a control, the production of cytokines in melanoma cells, H 1PV selleck chemical infected melanoma cells, and of immature and mature DC was measured. Mature DC controls were obtained by adding a cytokine cocktail as previously described. Supernatants were prepared by aspirating sellectchem Inhibitors,Modulators,Libraries media from the co culture and by diluting 1 1 with fresh medium. http://www.selleckchem.com/products/MG132.html Tumor necrosis factor a and Inhibitors,Modulators,Libraries interleukin 6 levels were deter mined by ELISA kits according to the manufacturers protocol. Results Cytotoxicity of H 1PV infected MZ7 Mel cells, expression of H 1PV proteins and virus driven transgenes The cytotoxic effect of H 1PV in MZ7 Mel cells was determined by time dependent measurement of cell growth.
H 1PV infection of MZ7 Mel cells with an MOI of 20 resulted in an approximately 50% reduction of cell growth at day 4 and 6 p.
Inhibitors,Modulators,Libraries i.
Six days p. i, cells Inhibitors,Modulators,Libraries continued to grow, but with a significantly reduced growth rate compared to controls. Inhibitors,Modulators,Libraries This may Inhibitors,Modulators,Libraries Inhibitors,Modulators,Libraries be due Inhibitors,Modulators,Libraries to the fact that a threshold of NS1 has to be present to induce cell cycle arrest. Until critical NS1 levels Inhibitors,Modulators,Libraries occur, cells can proliferate. Time dependent expression of the viral non structure protein NS1 was documented in MZ7 Mel cells by western blotting at 1 day p. i. with the highest expression levels found 2 days p. i. On day 6 p. i, NS1 expression decreased, most likely due to H 1 mediated induction of apoptosis.
Inhibitors,Modulators,Libraries Further results of the luciferase assay using parvovirus hH11600luc indicated Inhibitors,Modulators,Libraries a more than 200 fold induction of lucifer ase activity 3 days p. i.
compared Inhibitors,Modulators,Libraries Inhibitors,Modulators,Libraries with non infected cells. Luciferase activity was further enhanced 4 days p. i.
Thus data suggest that H 1PV induced Inhibitors,Modulators,Libraries cell kill ing was correlated with NS1. Analysis of DC activity phagocytosis and maturation The immunogenicity Inhibitors,Modulators,Libraries of tumor cells was determined by phagocytosis and the presentation of tumor antigens by DC. We therefore investigated the activation of DC following co culture with H 1PV induced MZ7 Mel lysates compared with a panel of control MZ7 Mel cell preparations. Phagocytosis of MZ7 Mel preparations by immature DC was quantified by flow cytometry.
The highest proportion of phagocytosing DC was 38%, detected after co incubation with TCLs from H 1PV infected MZ7 Mel 10 days p.
i, compared with 29% DC coincu bated with UV irradiated and ultrasonic treated tumor cells respectively, and 16% from untreated melanoma cells.
Thus, phagocytosis in immature DC was most effectively stimulated by H 1PV induced melanoma http://www.selleckchem.com/products/kpt-330.html cell lysates. As previously been shown, treatment of immature DC with a cocktail of pro inflammatory Trichostatin A mechanism cytokines led to DC maturation characterized by a major increase in CD80, CD83 and CD86 expression. In order to investigate whether H 1PV induced melanoma selleck kinase inhibitor cell lysates modulate DC maturation, the expression of these surface markers was analyzed following incubation with H 1PV induced melanoma cell lysates taken 10 days p. i.