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Systemic mastocytosis ( SM) is a rare myeloproliferative disease without curative therapy. Despite clinical variability, the majority of patients harbour a KIT- D816 V mutation, but efforts to inhibit mutant KIT with tyrosine kinase inhibitors have been unsatisfactory, indicating a need for new preclinical approaches to identify alternative targets and novel therapies in this disease. Murine models to date have been limited and do not fully recapitulate the most aggressive forms of SM. We describe the generation of a transgenic zebrafish model expressing the human KIT- D816 V mutation. Adult fish demonstrate a myeloproliferative disease phenotype, including features of aggressive SM in haematopoeitic tissues and high expression levels of endopeptidases, consistent with SM patients. Transgenic embryos demonstrate a cell-cycle phenotype with corresponding expression changes in genes associated with DNA maintenance and repair, such as reduced dnmt1. In addition, epcam was consistently downregulated in both transgenic adults and embryos. Decreased embryonic epcam expression was associated with reduced neuromast numbers, providing a robust in vivo phenotypic readout for chemical screening in KIT- D816 V-induced disease. This study represents the first zebrafish model of a mast cell disease with an aggressive adult phenotype and embryonic markers that could be exploited to screen for novel agents in SM. [ABSTRACT FROM AUTHOR]

Transcriptional silencing by CpG island methylation is a prevalent mechanism of tumor-suppressor gene suppression in cancers. Genetic experiments have defined the importance of the DNA methyltransferase Dnmt1 for the maintenance of methylation in mouse cells and its role in neoplasia. In human bladder cancer cells, selective depletion of DNMT1 with antisense inhibitors has been shown to induce demethylation and reactivation of the silenced tumor-suppressor gene CDKN2A. In contrast, targeted disruption of DNMT1 alleles in HCT116 human colon cancer cells produced clones that retained CpG island methylation and associated tumor-suppressor gene silencing, whereas HCT116 clones with inactivation of both DNMT1 and DNMT3B showed much lower levels of DNA methylation, suggesting that the two enzymes are highly cooperative. We used a combination of genetic (antisense and siRNA) and pharmacologic (5-aza-2′-deoxycytidine) inhibitors of DNA methyl transferases to study the contribution of the DNMT isotypes to cancer-cell methylation. Selective depletion of DNMT1 using either antisense or siRNA resulted in lower cellular maintenance methyltransferase activity, global and gene-specific demethylation and re-expression of tumor-suppressor genes in human cancer cells. Specific depletion of DNMT1 but not DNMT3A or DNMT3B markedly potentiated the ability of 5-aza-2′-deoxycytidine to reactivate silenced tumor-suppressor genes, indicating that inhibition of DNMT1 function is the principal means by which 5-aza-2′-deoxycytidine reactivates genes. These results indicate that DNMT1 is necessary and sufficient to maintain global methylation and aberrant CpG island methylation in human cancer cells. [ABSTRACT FROM AUTHOR]

Antisense-based molecules targeting HIV-1 RNA have the potential to be used as part of gene or drug therapy to treat HIV-1 infection. In this study, HIV-1 RNA was screened to identify more conserved and accessible target sites for ribozymes based on the hepatitis delta virus motif. Using a quantitative screen for effects on HIV-1 production, we identified a ribozyme targeting a highly conserved site in the Gag coding sequence with improved inhibitory potential compared to our previously described candidates targeting the overlapping Tat/Rev coding sequence. We also demonstrate that this target site is highly accessible to short hairpin directed RNA interference, suggesting that it may be available for the binding of antisense RNAs with different modes of action. We provide evidence that this target site is structurally conserved in diverse viral strains and that it is sufficiently different from the human transcriptome to limit off-target effects from antisense therapies. We also show that the modified hepatitis delta virus ribozyme is more sensitive to a mismatch in its target site compared to the short hairpin RNA. Overall, our results validate the potential of a new target site in HIV-1 RNA to be used for the development of antisense therapies.

Phosphodiesterase 4 (PDE4) inhibitors elevate cyclic adenosine 5'-monophosphate (cAMP), and this elevation has been shown to inhibit inflammatory cytokines such as tumor necrosis factor-alpha (TNF-alpha). Using TNF-alpha as a biomarker, we have developed transcription-based assays to examine inhibition of PDE4 activity in human and guinea pig whole blood. In vitro inhibition by PDE4 inhibitors was measured using quantitative PCR (qPCR) analysis of TNF-alpha mRNA levels in whole blood stimulated with lipopolysaccharide (LPS). The kinetics of human TNF-alpha mRNA production were analyzed and shown to be highest 4 hr following LPS stimulation. The guinea pig displayed kinetics of TNF-alpha transcription similar to those of the human. Analysis of inhibition of human TNF-alpha protein production was performed by immunoassay and shown to correlate with inhibition of transcription for three of the four compounds tested. Roflumilast was found to be 9-fold more potent for TNF-alpha inhibition in the qPCR assay than in the protein assay. The potencies of L-826,141 and roflumilast were determined in human and guinea pig whole blood by qPCR, with IC(50) values of 270 and 20 nM, respectively, in humans and 100 and 10 nM, respectively, in guinea pigs. These results show that the potency of PDE4 inhibitors can be monitored in whole blood using a transcription-based assay, and that this type of assay can be adapted to various species provided the TNF-alpha nucleotide sequence is known. The in vitro whole blood IC(50) for TNF-alpha inhibition was compared to inhibition in the ovalbumin-challenged guinea pig model of bronchoconstriction. Obtaining plasma levels at the IC(50) determined in vitro for L-826,141 and roflumilast provides significant inhibition of bronchoconstriction. This suggests that TNF-alpha can be used as a whole blood biomarker in the guinea pig for PDE4 inhibition in this inflammatory model.