Synthetic oligonucleotides (ODN) expressing repetitive TTAGGG motifs patterned after hexameric sequences present at high frequency in mammalian teleomeres down-regulate the inflammatory immune responses elicited by a broad range of TLR ligands and the adaptive immune cell responses induced by polyclonal activators and antigens. These suppressive ODN are useful in the treatment of diseases characterized by over-exuberant immune responses, including septic shock and autoimmunity. Since joining the NCI we demonstrated that suppressive ODN are also useful in the prevention/treatment of the life-threatening inflammation caused by silicosis. Specifically, suppressive ODN significantly reduced mortality and morbidity in a murine silicosis model. We are in the process of examining whether susceptibility to chronic silica-induced inflammation and pulmonary tumors can also be reduced by early Rx with suppressive ODN. Recent results suggest that systemically administered suppressive ODN can alter the host immune milieu. We are harnessing this effect to reduce host susceptibility to inflammation-induced cancer. The major focus of our ongoing research involves examining the effect of Sup ODN in the DMBA/TPA model of skin carcinogenesis. We find that Sup ODN significantly reduce both the number of mice that develop papillomas and the number of papillomas/animal in this murine model of inflammation-associated tumorigenesis. Dose-response studies established that 50 ug of Sup ODN reduced the incidence of papillomas and the number of papillomas/animal by 90 - 95% (p = 0.027), with lower doses being progressively less effective. Sup ODN reduced papilloma formation when delivered either locally or systemically, whereas control ODN had no effect on papilloma formation. To clarify the mechanism by which Sup ODN inhibit papilloma formation, various parameters of TPA induced inflammation were examined. TPA causes cutaneous edema, leukocyte infiltration and triggers a 3 - 18 fold increase in mRNA encoding chemokines and other mediators of inflammation (including CXCL2, CCL2, ODC and COX-2). The administration of Sup ODN significantly reduced all measures of inflammation, whereas control ODN had no significant effect. The proliferation of established papillomas is maintained by the inflammatory milieu that persists even after TPA treatment ceases, leading to a >3-fold increase in papilloma size over 3 months. Papillomas elicited by DMBA/TPA were treated with Sup ODN starting 4 wk after the cessation of the TPA (at week 20). This late administration of Sup ODN prevented the further proliferation of established papillomas, whereas control ODN had no effect (p. <.01). These results demonstrate that the continued growth as well as the initial development of inflammation-associated tumors may be inhibited by Sup ODN. We are using microarray technology to identify the genes and regulatory networks triggered by suppressive ODN. These microarray studies indicate that very large numbers of genes are rapidly down-regulated following the administration of suppressive ODN. Two mechanisms by which Sup ODN broadly reduce gene expression are the focus of ongoing evaluation: i) that Sup ODN target TTAGGG and/or CCCTAA motifs present in the regulatory regions of critical genes (such as MAPKs) and ii) that Sup ODN inhibit the activity of bZIP proteins (regulatory DNA binding proteins that share a basic leucine zipper domain). In the context of MAPK regulation, mRNA encoding MAPK1, MAPK3 and MAPK14 (which stimulate the ERK-2, ERK-3 and p38 dependent pathways, respectively), as well as the transcription factors they regulate (ATF2, CREB1, NFKB1), contain TTAGGG and/or CCCTAA motifs in their regulatory regions that could be targeted by Sup ODN via an anti-sense mechanism . Since MAPKs exert a stabilizing effect on mRNAs encoding multiple inflammatory genes, down-regulating their expression could have a broad effect on the innate immune response. bZIP proteins are transcription factors that broadly influence gene expression. Transcription binding site analysis shows that many genes down-regulated by Sup ODN contain regulatory domains recognized by bZIP proteins (e.g. CREB1, CEBPA, and FOS). Indeed, 79% of the components in the TLR9 signaling pathway regulated by bZIP proteins are significantly down-regulated by Sup ODN (p <.0001). We examined whether Sup ODN inhibited the binding of bZIP proteins to their target DNA sequences and found that Sup ODN selectively inhibited the binding of three different classes of bZIP protein (CREB, C/EBPa and MAFg) to their target DNAs. Information gathered on the targets and mechanism(s) of action of suppressive ODN will support studies designed to explore their therapeutic utility.
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