Adult cancers may derive from stem or early progenitor cells 1,2 . Epigenetic modulation of gene expression is essential for normal function of these early cells, but is highly abnormal in cancers, which often exhibit aberrant promoter CpG island hypermethylation and transcriptional silencing of tumor suppressor genes and pro-differentiation factors [3][4][5] . We find that, for such genes, both normal and malignant embryonic cells generally lack the gene DNA hypermethylation found in adult cancers. In embryonic stem (ES) cells, these genes are held in a "transcription ready" state mediated by a "bivalent" promoter chromatin pattern consisting of the repressive polycomb group (PcG) H3K27me mark plus the active mark, H3K4me. However, embryonic carcinoma (EC) cells add two key repressive marks, H3K9me2 and H3K9me3, both associated with DNA hypermethylated genes in adult cancers [6][7][8] . We hypothesize that cell chromatin patterns and transient silencing of these important growth regulatory genes in stem or progenitor cells of origin for cancer may leave these genes vulnerable to aberrant DNA hypermethylation and heritable gene silencing in adult tumors.Correspondence may be addressed to S.B.B. at sbaylin@jhmi.edu. Competing Interests Statement. The commercial rights to the MSP technique belong to Oncomethylome. S.B.B and J.G.H. serve as consultants to Oncomethylome and is entitled to royalties from any commercial use of this procedure. Epigenetic gene silencing and associated promoter CpG island DNA hypermethylation are prevalent in all cancer types, and provide an alternative mechanism to mutations by which tumor suppressor genes may be inactivated within a cancer cell [3][4][5] . These epigenetic changes may precede genetic changes in pre-malignant cells and foster the accumulation of additional genetic and epigenetic hits 9 . Adult cancers may derive from stem or early progenitor cells 1, 2 , and epigenetic modulation of gene expression is essential for normal function of these early cells. We now explore whether DNA hypermethylation and heritable silencing of groups of genes in adult tumor initiation and progression might reflect chromatin properties for these genes associated with a stem or precursor cell of origin. NIH Public AccessWe compared the epigenetic status of a group of genes frequently hypermethylated and silenced in adult cancers ( Fig. 1-all (Fig. 1). Among the genes studied, 13 of 29 (45%) are hypermethylated in a single line, HCT-116, of adult colon cancer, but none are hypermethylated in ES cells, and only 3% and 7% were completely methylated in the Tera-1 and Tera-2 EC lines, respectively. Thus, the key epigenetic parameter of promoter CpG island hypermethylation which is common in a large group of genes in adult cancer cells does not seem to be a common feature of EC cells.In murine ES cells, many developmental genes are maintained in a state of low transcriptional activity and are available for transcription increases or decreases when differentiation cues are received 11 . Our s...

Traditional approaches to the preclinical investigation of cancer therapies rely on the use of established cell lines maintained in serum-based growth media.

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Nonsteroidal antiinf lammatory drugs reduce the risk of colon cancer, possibly via cyclooxygenase (COX) inhibition. The growth factor-inducible COX-2, which is overexpressed in neoplastic colonic tissue, is an attractive target to mediate this effect. Herein we have exploited the ability of a human colon cancer cell line, HCA-7 Colony 29, to polarize when cultured on Transwell (Costar) filters to study COX-2 production and the vectorial release of prostaglandins (PGs). Administration of type ␣ transforming growth factor to the basolateral compartment, in which the epidermal growth factor receptor (EGFR) resides, results in a marked induction of COX-2 immunoreactivity at the base of the cells and the unexpected appearance of COX-2 in the nucleus. The increase in COX-2 protein is associated with a dose-and time-dependent increase in PG levels in the basolateral, but not apical, medium. Amphiregulin is the most abundantly expressed EGFR ligand in these cells, and the protein is present at the basolateral surface. EGFR blockade reduces baseline COX-2 immunoreactivity, PG levels, and mitogenesis in a concentration-dependent manner. Two specific COX-2 inhibitors, SC-58125 and NS 398, also, in a dose-dependent manner, attenuate baseline and type ␣ transforming growth factor-stimulated mitogenesis, although PG levels are decreased >90% at all concentrations of inhibitor tested. These findings show that activation of the EGFR stimulates COX-2 production and its translocation to the nucleus, vectorial release of PGs, and mitogenesis in polarized HCA-7 Colony 29 cells.In the gastrointestinal tract, prostaglandins (PGs) mediate important functions, including motility, vascular tone, angiogenesis, mucosal protection, and immune responsiveness (1). Inasmuch as epithelial cells are capable of PG synthesis, it is feasible that PGs synthesized in the gastrointestinal epithelium regulate these functions by paracrine pathways in response to luminal or serosal stimuli. Although data exist in support of vectorial release of PGs in the isolated rat colon (2), as well as in other tissues and polarized kidney-derived cells (3-5), regulatory mechanisms have not been defined more precisely.Cyclooxygenases (COXs) are key enzymes in the conversion of arachidonic acid (AA) to PGs and other eicosanoids. Two isoforms of the enzyme have been characterized. COX-1 in most cells is expressed constitutively, and a second inducible form known as COX-2 has been identified (refs. 6-8; for review see ref. 9). Recent observations indicate that many colonic polyps and cancers overexpress COX-2 (10-12) and that inhibition of this enzyme by nonsteroidal antiinflammatory drugs decreases the risk of colonic neoplasia (13-20), emphasizing the importance of defining potential autocrine and paracrine pathways for regulation of gastrointestinal epithelial growth by COX. Signaling through the epidermal growth factor receptor (EGFR) induces COX-2 expression, and unregulated overexpression of COX-2 results in a tumorigenic phenotype in the rat intestinal e...

Bcl-2 is a central regulator of cell survival that is overexpressed in the majority of small cell lung cancers (SCLC) and contributes to both malignant transformation and therapeutic resistance. We compared primary SCLC xenografts prepared from de novo human tumors with standard cell line-based xenografts in the evaluation of a novel and highly potent small molecule inhibitor of Bcl-2, ABT-737. ABT-737 induced dramatic regressions in tumors derived from some SCLC cell lines. In contrast, only one of three primary xenograft SCLC tumors showed significant growth inhibition with ABT-737. Explanations for this apparent dichotomy may include relatively low expression of Bcl-2 in the primary xenografts or inherent differences in the model systems. The addition of etoposide to ABT-737 in the primary xenografts resulted in significant decreases in tumor growth, underscoring the clinical potential of ABT-737 in combination therapy. To identify factors that may contribute to resistance to ABT-737 and related inhibitors, we isolated resistant derivatives of an initially sensitive cell line-based xenograft. Acquired resistance in this model was associated with decreases in the expression of the primary target Bcl-2, of proapoptotic partners of Bcl-2 (Bax and Bim), and of Bcl-2:Bim heterodimers. Expression profiling reveals 85 candidate genes demonstrating consistent changes in gene expression with acquired resistance. Taken together, these data have specific implications for the clinical development of Bcl-2 inhibitors for SCLC and broader implications for the testing of novel anticancer strategies in relevant preclinical models. [Cancer Res 2008;68(7):2321-8]

Previous studies have shown that IL-10 may modulate immune responses towards the humoral arm by inhibiting production of cytokines involved in cell-mediated responses. In the present studies, we found that mRNA to IL-10 could be demonstrated in 66% of melanoma cell lines by PCR amplification of reverse-transcribed mRNA and in supernatants of the cell lines by ELISA. Release into the supernatants increased approximately 2-fold each day up to 3 days. The MW of 35S-labelled IL-10 secreted by melanoma cells was similar to that reported in previous studies. In the present studies we also examined whether IL-10 may be responsible for some of the immunosuppressive effects of the melanoma cell supernatants observed in previous studies, by testing whether MAbs against IL-10 could reverse the inhibitory effects of these supernatants. Recombinant IL-10 and melanoma supernatants were found to inhibit production of TNF-alpha, IFN-gamma, IL-2 and mixed lymphocyte reactions but reversal of these effects of melanoma supernatants by MAbs against IL-10 was only seen in the case of TNF-alpha production. These results extend the range of cell types known to produce IL-10 and indicate that malignancy of certain cell types may lead to unregulated production of IL-10 that could have the potential to modulate immune responses against the tumor.

The isoprostanes (IsoPs) are novel bioactive prostaglandin-like compounds produced in vivo by free radical-catalyzed peroxidation of arachidonyl-containing lipids. Previously, we have identified IsoPs containing F-type and D- and E-type prostane rings that are formed by reduction and rearrangement of IsoP endoperoxide intermediates, respectively. We now explore whether thromboxane B2 (TxB2)-like compounds, termed B2-isothromboxanes (B2-IsoTxs), are formed by rearrangement of IsoP endoperoxides. Detection of these compounds was carried out using a stable isotope dilution mass spectrometric assay originally developed for quantification of cyclooxygenase-derived TxB2. Incubations of arachidonic acid with Fe/ADP/ascorbate for 30 min in vitro generated a series of peaks representing putative B2-IsoTx at levels of 62.4 +/- 21.0 ng/mg arachidonate. Using various chemical modification and derivatization approaches, it was determined that these compounds contained hemiacetal ring structures and two double bonds, as would be expected for B2-IsoTx. Analysis of the compounds by electron ionization mass spectrometry yielded multiple mass spectra similar to those of TxB2. B2-IsoTxs are also formed esterified to phospholipids; oxidation of arachidonyl-containing phosphatidylcholine in vitro followed by hydrolysis resulted in the release of large amounts of these compounds. To explore whether B2-IsoTxs are also formed in vivo, a well characterized animal model of lipid peroxidation consisting of orogastric administration of CCl4 to rats was used. Levels of B2-IsoTx esterified in lipids in the liver increased 41-fold from 2.5 +/- 0.5 to 102 +/- 30 ng/g of liver. In addition, circulating levels of free compounds increased from undetectable (<5 pg/ml) to 185 +/- 30 pg/ml after CCl4, a 37-fold increase. Thus, we have provided evidence that IsoTxs constitute another novel class of eicosanoids produced in vivo nonenzymatically by free radical-catalyzed lipid peroxidation. These studies thus expand our understanding of products of lipid peroxidation formed in vivo from the free radical-catalyzed peroxidation of arachidonic acid.

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