Dendritic cell-mediated survival signals in Eµ-Myc B-cell lymphoma depend on the transcription factor C/EBPß.

The capacity of dendritic cells (DCs) to regulate tumour-specific adaptive immune responses depends on their proper differentiation and homing status. Whereas DC-associated tumour-promoting functions are linked to T-cell tolerance and formation of an inflammatory milieu, DC-mediated direct effects on tumour growth have remained unexplored. Here we show that deletion of DCs substantially delays progression of Myc-driven lymphomas. Lymphoma-exposed DCs upregulate immunomodulatory cytokines, growth factors and the CCAAT/enhancer-binding protein ß (C/EBPß). Moreover, Eµ-Myc lymphomas induce the preferential translation of the LAP/LAP* isoforms of C/EBPß. C/EBPß(-/-) DCs are unresponsive to lymphoma-associated cytokine changes and in contrast to wild-type DCs, they are unable to mediate enhanced Eµ-Myc lymphoma cell survival. Antigen-specific T-cell proliferation in lymphoma-bearing mice is impaired; however, this immune suppression is reverted by the DC-restricted deletion of C/EBPß. Thus, we show that C/EBPß-controlled DC functions are critical steps for the creation of a lymphoma growth-promoting and -immunosuppressive niche.

Epigenetic regulation of CD133/PROM1 expression in glioma stem cells by Sp1/myc and promoter methylation.

Tumor stem cells, postulated to be the source cells for malignancies, have been identified in several cancers using cell-surface expression of markers including CD133, a pentaspan membrane protein. CD133+ve cells form neurospheres, exhibit self-renewal and differentiation, and are tumorigenic. However, despite its association with stem cells, a causal relationship of CD133 to tumorigenesis remains to be defined. Hypothesizing that specific epigenetic and transcription factors implicated in driving the stem cell state may concurrently regulate CD133 expression in stem cells, we analyzed the structure and regulation of CD133 promoter in glioma stem cells and glioma cell lines. Initially, a minimal promoter region was identified by analyzing the activity of CD133 promoter-driven luciferase-expressing 5'-and 3'-deletion-constructs upstream of the transcription start site. This region contained a CpG island that was hypermethylated in CD133-ve glioma stem cells (GSC) and glioma cells but unmethylated in CD133+ve ones. Of several predicted TF-binding sites in this region, the role of tandem Sp1 (-242 and -221) and two Myc (-541 and -25)-binding sites were examined. Overexpression of Sp1 or Myc increased CD133 minimal promoter-driven luciferase activity and CD133 levels in GSC and in glioma cell line. Mithramycin, a Sp1 inhibitor, decreased minimal promoter activity and downregulated CD133 levels in GSC. Gel-shift assays demonstrated direct binding of Sp1 to their predicted sites that was competitively inhibited by oligonucleotide-binding-site sequences and supershifted by anti-Sp1 confirming the interaction. Sp1 and Myc-antibody chromatin immunoprecipitation (ChIP) analysis in GSC showed enrichment of regions with Sp1 and Myc-binding sites. In CD133-ve cells, ChIP analysis showed binding of the methyl-DNA-binding proteins, MBD1, MBD2 and MeCP2 to the methylated CpG island and repression of transcription. These results demonstrate that Sp1 and Myc regulate CD133 transcription in GSC and that promoter methylation and methyl-DNA-binding proteins cause repression of CD133 by excluding transcription-factor binding.

The effect of H-ras expression on tumorigenicity and immunogenicity of Balb/c 3T3 fibroblasts.

In an attempt to define immunological parameters affected by the H-ras oncogene, we have used Balb/c 3T3 cells transfected with either H-ras (98/6), H-ras+v-myc (98/4v) or plasmid only (98/1). We found that while control and oncogene-transfected Balb/c 3T3 cells exhibit similar low sensitivity to lysis by natural killer (NK) cells, H-ras+v-myc-transfected cells could immunize syngeneic Balb/c mice and induce cytotoxic T cells (CTL) with broad specificity, that lysed all types of Balb/c 3T3 cells tested. Immunization of Balb/c mice with 98/4v cells prevented homologous tumor formation and partially inhibited the formation of tumors derived from H-ras-transfected cells. 98/6 cells were not immunogenic in vivo and did not protect the animals from a challenge of 98/6 cells. The results suggested that CTLs but not NK effector cells were important for eliciting in vivo tumor rejection of H-ras+v-myc-transfected cells. In contrast, antigens eliciting the cytotoxic T-cell response, and possibly also the in vivo tumor cell rejection response, were expressed on all cell types tested but were immunogenic only on the surface of 98/4v cells. We further determined major histocompatibility complex (MHC) class-I molecule expression on the outer cell surface and found that H-2K was down-regulated in H-ras-transfected cells. The results support the observation that oncogenes can down-regulate specific MHC antigens, thereby preventing presentation of tumor antigens and allowing tumor escape from immune recognition.