Naturally occurring regulatory T cells (T reg cells) are a thymus-derived subset of T cells, which are crucial for the maintenance of peripheral tolerance by controlling potentially autoreactive T cells. However, the underlying molecular mechanisms of this strictly cell contact–dependent process are still elusive. Here we show that naturally occurring T reg cells harbor high levels of cyclic adenosine monophosphate (cAMP). This second messenger is known to be a potent inhibitor of proliferation and interleukin 2 synthesis in T cells. Upon coactivation with naturally occurring T reg cells the cAMP content of responder T cells is also strongly increased. Furthermore, we demonstrate that naturally occurring T reg cells and conventional T cells communicate via cell contact–dependent gap junction formation. The suppressive activity of naturally occurring T reg cells is abolished by a cAMP antagonist as well as by a gap junction inhibitor, which blocks the cell contact–dependent transfer of cAMP to responder T cells. Accordingly, our results suggest that cAMP is crucial for naturally occurring T reg cell–mediated suppression and traverses membranes via gap junctions. Hence, naturally occurring T reg cells unexpectedly may control the immune regulatory network by a well-known mechanism based on the intercellular transport of cAMP via gap junctions.

Ewing tumors (ET) are highly malignant, localized in bone or soft tissue, and are molecularly defined by ews/ets translocations. DNA microarray analysis revealed a relationship of ET to both endothelium and fetal neural crest. We identified expression of histone methyltransferase enhancer of Zeste,Drosophila, Homolog 2 (EZH2) to be increased in ET. Suppressive activity of EZH2 maintains stemness in normal and malignant cells. Here, we found EWS/FLI1 bound to the EZH2 promoter in vivo, and induced EZH2 expression in ET and mesenchymal stem cells. Down-regulation of EZH2 by RNA interference in ET suppressed oncogenic transformation by inhibiting clonogenicity in vitro. Similarly, tumor development and metastasis was suppressed in immunodeficient Rag2^−/−γ_C^−/−mice. EZH2-mediated gene silencing was shown to be dependent on histone deacetylase (HDAC) activity. Subsequent microarray analysis of EZH2 knock down, HDAC-inhibitor treatment and confirmation in independent assays revealed an undifferentiated phenotype maintained by EZH2 in ET. EZH2 regulated stemness genes such as nerve growth factor receptor (NGFR), as well as genes involved in neuroectodermal and endothelial differentiation (EMP1, EPHB2, GFAP, and GAP43). These data suggest that EZH2 might have a central role in ET pathology by shaping the oncogenicity and stem cell phenotype of this tumor.

The product of the proto‐oncogene c‐myc (myc) is a potent activator of cell proliferation. In Burkitt lymphoma (BL), a human B‐cell tumor, myc is consistently found to be transcriptionally activated by chromosomal translocation. The mechanisms by which myc promotes cell cycle progression in B‐cells is not known. As a model for myc activation in BL cells, we have established a human EBV‐EBNA1 positive B‐cell line, P493‐6, in which myc is expressed under the control of a tetracycline regulated promoter. If the expression of myc is switched off, P493‐6 cells arrest in G0/G1 in the presence of serum. Re‐expression of myc activates the cell cycle without inducing apoptosis. myc triggers the expression of cyclin D2, cyclin E and Cdk4, followed by the activation of cyclin E‐associated kinase and hyper‐phosphorylation of Rb. The transcription factor E2F‐1 is expressed in proliferating and arrested cells at constant levels. The Cdk inhibitors p16, p21, p27 and p57 are expressed at low or not detectable levels in proliferating cells and are not induced after repression of myc. Ectopic expression of p16 inhibits cell cycle progression. These data suggest that myc triggers proliferation of P493‐6 cells by promoting the expression of a set of cell cycle activators but not by inactivating cell cycle inhibitors. Int. J. Cancer 87:787–793, 2000.

Ewing family tumors (EFTs) are small round blue cell tumors that show features of neuroectodermal differentiation. However, the histogenetic origin of EFTs is still a matter of debate. We used high-density DNA microarrays for the identification of EFT-specific gene expression profiles in comparison with normal tissues of diverse origin. We identified 37 genes that are up-regulated in EFTs compared with normal tissues and validated expression of these genes in EFTs by both conventional and quantitative reverse transcription-polymerase chain reaction. The expression pattern of EFT-associated genes in normal tissues indicated a high similarity between EFTs and fetal and neuronal as well as endothelial tissues and supports the concept that a primitive neural crest-derived progenitor at the transition to mesenchymal and endothelial differentiation is transformed in EFTs. EFT-associated genes could be used for molecular discrimination between EFTs and other small round blue cell tumors and clearly identified a cell line (SK-N-MC) that was initially established as neuroblastoma as being an EFT. Ectopic expression of the EFT-specific EWS-FLI1 fusion protein in human embryonic kidney (HEK293) cells was not sufficient to induce the complete EFT-specific gene expression signature, suggesting that the EFT-specific gene expression profile is not just a consequence of EWS-FLI1 expression but depends on the histogenetic background of the EFT stem cell.

Chromatin modifications are increasingly recognized as a key mechanism in cancer. The histone methyltransferase Enhancer of Zeste, Drosophila, Homolog 2 (EZH2), the enzymatic subunit of the polycomb PRC2 complex methylates histone H3K27, thereby, mediating gene silencing. EZH2 is overexpressed in a variety of tumor tissue including breast and prostate. Ewing tumors (ET), alias peripheral neuroectodermal tumors (PNET), are highly malignant tumors molecularly defined by ews/ets translocations. We found EWS-FLI1 bound to the EZH2 promoter in vivo. Other components of the PRC2 complex, like EED or SUZ12 were not deregulated in ET. Downregulation of EZH2 by RNA interference suppressed tumor development and metastasis in vivo and microarray analysis of EZH2 knock down revealed an EZH2-maintained, undifferentiated, reversible phenotype in ET. EZH2 suppression resulted in a generalized loss of H3K27me3 as well as increase in H3 acetylation. ChIP-Chip assays for H3K27me3 verified such genes that had specifically lost H3K27me3 upon EZH2 silencing, suggesting that stemness features are preserved via epigenetic mechanisms. Taken together, the genetic EWS-FLI1 translocation is intimately linked to global and gene specific epigenetic alterations in ET biology. EZH2 mediates neuroectodermal and endothelial embryonal tumor stem cell growth and metastatic spread induced by a translocation derived chimeric transcription factor.