Background We reported that DNA-PK is critical for the expression of NF-κB-dependent genes in TNF-α-treated glioblastoma cells, suggesting an involvement in inflammatory diseases. Objective To investigate the role of DNA-PK in asthma. Methods Cell culture and ovalbumin or house dust mite (HDM)-based murine asthma models were used in this study. Results DNA-PK was essential for monocyte adhesion to TNF-α–treated-endothelial cells. Administration of the DNA-PK inhibitor, NU7441, reduced airway eosinophilia, mucus hypersecretion, airway hyperresponsiveness (AHR), and OVA-specific IgE production in mice that were pre-challenged with ovalbumin. Such effects correlated with a marked reduction in lung VCAM-1 expression and production of several cytokines, including IL-4/IL-5/IL-13/eotaxin/IL-2/IL-12 and the chemokines MCP-1/KC with negligible effect on IL-10/IFN-γ production. DNA-PK inhibition, by gene heterozygosity, also prevented manifestation of asthma-like traits. These results were confirmed in a chronic model of asthma using HDM, a human allergen. Remarkably, such protection occurred without causing SCID. Adoptive transfer of Th2-skewed OT-II-WT CD4+ T cells reversed IgE and Th2 cytokine production but not AHR in ovalbumin-challenged DNA-PKcs+/− mice. DNA-PK inhibition reduced IL-4/IL-5/IL-13/eotaxin/IL-8/MCP-1 production without affecting IL-2/IL-12/IFN-γ/IP-10 production in CD3/CD28-stimulated human CD4+T cells potentially by blocking expression of gata-3. These effects occurred without significant reductions in T-cell proliferation. In mouse CD4+T cells, DNA-PK inhibition, in vitro, severely blocked C3/CD28-induced gata-3 and t-bet expression in CD4+T cells and prevented differentiation of Th1 and Th2 cells under respective Th1- and Th2-skewing conditions. Conclusion Our results suggest DNA-PK as a novel determinant of asthma and a potential target for the treatment of the disease.
The present study establishes poly(ADP-ribose)polymerase's (PARP's) role in chronic asthma, demonstrates that it is activated in human asthma, increases the clinical relevance of targeting PARP for blocking or preventing chronic asthma in humans and presents olaparib as a likely candidate drug.
PDZ domain containing 1 (PDZK1) is a scaffold protein that plays a role in the fate of several proteins. Estrogen can induce PDZK1 gene expression; however, our recent report showed that PDZK1 expression in the breast cancer cell line MCF-7 is indirect and involves insulin-like growth factor (IGF)-1 receptor function. Such a relationship was established in cell culture systems and human breast cancer tissues. Here we show that overexpression of PDZK1 promoted an increase in cyclin D1 and enhanced anchorageindependent growth of MCF-7 cells in the absence of 17β-estradiol, suggesting that PDZK1 harbors oncogenic activity. Indeed, PDKZ1 overexpression enhanced epidermal growth factor receptor (EGFR)-stimulated MEK/ERK1/2 signaling and IGF-induced Akt phosphorylation. PDZK1 appeared to play this role, in part, by stabilizing the integrity of the growth promoting factors Akt, human epidermal growth factor receptor 2 (Her2/Neu) and EGFR. Increased Akt levels occurred via a decrease in the ubiquitination of the kinase. PDZK1 overexpression was associated with resistance to paclitaxel/5-fluorouracil/etoposide only at low concentrations. Although the increased stability of Akt was sensitive to heat shock protein 90 (HSP90) inhibition, increased levels of the cochaperone cell division cycle 37 (Cdc37), as well as its ability to bind PDZK1, appear to play a larger role in kinase stability. Using human tissue microarrays, we show strong positive correlation between PDZK1, Akt and Cdc37 protein levels, and all correlated with human breast malignancy. There were no positive correlations between PDZK1 and Cdc37 at the mRNA levels, confirming our in vitro studies. These results demonstrate a relationship between PDZK1, Akt and Cdc37, and potentially Her2/Neu and EGFR, in breast cancer, representing a new axis that can be targeted therapeutically to reduce the burden of human breast cancer.
We previously showed that DNA fragmentation factor, which comprises a caspase-3-activated DNase (CAD) and its inhibitor (ICAD), may influence the rate of cell death by generating PARP-1-activating DNA breaks. Here we tested the hypothesis that ICAD-deficient colon epithelial cells exhibiting resistance to death stimuli may accumulate additional genetic modifications, leading to a tumorigenic phenotype. We show that ICAD deficiency may be associated with colon malignancy in humans. Indeed, an examination of ICAD expression using immunohistochemistry in an array of both colon cancer and normal tissues revealed that ICAD expression levels were severely compromised in the cancerous tissues. Upon DNA damage caused by a low dose of irradiation, ICAD cells acquire a tumorigenic phenotype. Colon epithelial cells derived from ICAD mice showed a significant resistance to death induced by the colon carcinogen dimethylhydrazine in vitro and in mice. Such resistance was associated with a decrease in PARP-1 activation. In an animal model of dimethylhydrazine-induced colon tumorigenesis, ICAD−/− mice developed significantly higher numbers of tumors with markedly larger sizes than the wild-type counterparts. Interestingly, the phenotype of the ICAD−/− mice was not associated with a significant increase in the precancerous aberrant crypt foci suggesting a potential link to tumor progression rather than initiation. More importantly, ICAD deficiency was associated with severe genomic instability as assessed by array comparative genomic hybridization. Such genomic instability consisted most prominently of amplifications but with sizable deletions as compared to the wild-type counterparts affecting several cancer-related genes including RAF-1, GSN, LMO3, and Fzd6 independently of p53. Altogether, our results present a viable case for the involvement of ICAD deficiency in colon carcinogenesis and show that apoptosis and genomic instability may comprise the means by which such deficiency may contribute to the process of increasing susceptibility to carcinogen-induced tumorigenesis.
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