Fig. 4b demonstrates that when the pcDNA4/TO/CCL21 plasmid was click here tested following bisulfite conversion, PCR reactions with both primers produced a product indicating that the original plasmid DNA was

not methylated. In contrast, when DNA was extracted from two excised TRAMPC2/CCL21-L2 tumors (M1 and M2, Fig. 3a), both promoters appeared to be methylated, however, when a clonal outgrowth derived from tumor M1 was tested, PCR products formed with both primers suggesting that the section of tumor excised MG-132 chemical structure for clonal expansion had a functional promoter (not methylated). These data indicate that during tumor growth in the prostate gland, the promoter is variably methylated. Thus, in some sections of the tumor, the promoter may still be functional. CBL-0137 in vitro This may explain

why we detected some low-grade induction of CCL21 in some clonal lines derived from explants of TRAMPC2/TR/CCL21-L2 tumors (Fig. 3a, right panel). Fig. 4 The CCL21 transgene is retained but the CMV promoter is methylated in TRAMPC2/TR/CCL21 tumor cells following progressive tumor growth in vivo. a DNA extracted from cloned cell lines derived from TRAMPC2/TR/CCL21-L2 tumors were tested for the transgene by PCR using primers specific for CCL21 transgene. Lanes 1–7 represent PCR products obtained when DNA was extracted from cell lines derived from 7 different TRAMPC2/TR/CCL21-L2 tumors (Fig. 3a-left panel). PcDNA4/TO/CCL21 plasmid used for transfection was included as a positive control (lane 8) and mouse DNA was used as negative control (lane 9). b DNA extracted directly from TRAMPC2/TR/CCL21-L2 tumors (M1 and M2) and a cell line derived from tumor Pyruvate dehydrogenase lipoamide kinase isozyme 1 M1 (line M1.2, see Fig. 3a-right panel) were tested for methylation status of CCL21 transgene promoter (CMV). TO/CCL21 plasmid was used as negative control. Extracted DNA was bisulfite treated and then was used in two different PCR reactions using oligos 1 (directed against a region of the CMV promoter not containing methylation sites) or oligos 2 (directed against a region of the CMV promoter which contains methylation sites) Discussion In this report we showed that

TRAMP tumors were infiltrated with small population of DCs. Although expression of CD11c on intratumoral DCs was low relative to splenic DCs, it still exceeded the isotype control (Fig. 1). We also demonstrated that DCs infiltrating TRAMPC2 tumors had low levels of MHCII, B7.2 and CD40 expression compared to their normal splenic counterparts. Most of the intratumoral DCs were myeloid-derived because they displayed a CD8α− phenotype. In addition to DC infiltrate, TRAMP tumors were infiltrated primarily by macrophages and immature (Gr-1+) myeloid cells but few T and B cells. Because myeloid cells have been shown to be immunosuppressive in several tumor models [19, 20], we transfected TRAMPC2 tumors with CCL21, a chemoattractant for DCs and T cells.