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Immunohistochemistry for ZBP-89 and Ki67 in WT and VC:Zfp148F/F mice

Percentage of APC, CTNNB1 and ZNF148 mutations in CRC (TCGA)

In colorectal cancer, APC-mediated induction of unregulated cell growth involves posttranslational mechanisms that prevent proteasomal degradation of proto-oncogene β-catenin (CTNNB1) and its eventual translocation to the nucleus. However, about 10% of colorectal tumors also exhibit increased CTNNB1 mRNA. Here, we show in colorectal cancer that increased expression of ZNF148, the gene coding for transcription factor ZBP-89, correlated with reduced patient survival. Tissue arrays showed that ZBP-89 protein was overexpressed in the early stages of colorectal cancer. Conditional deletion of Zfp148 in a mouse model of Apc-mediated intestinal polyps demonstrated that ZBP-89 was required for polyp formation due to induction of Ctnnb1 gene expression. Chromatin immunoprecipitation (ChIP) and EMSA identified a ZBP-89–binding site in the proximal promoter of CTNNB1. Reciprocally, siRNA-mediated reduction of CTNNB1 expression also decreased ZBP-89 protein. ChIP identified TCF DNA binding sites in the ZNF148 promoter through which Wnt signaling regulates ZNF148 gene expression. Suppression of either ZNF148 or CTNNB1 reduced colony formation in WNT-dependent, but not WNT-independent cell lines. Therefore, the increase in intracellular β-catenin protein initiated by APC mutations is sustained by ZBP-89–mediated feedforward induction of CTNNB1 mRNA. Cancer Res; 76(23); 6877–87. ©2016 AACR.

Supplemental Methods, Figure Legends and References for Supplemental Methods

ZBP-89 Probe Sets:BFCOL1 and BERF1

In colorectal cancer, APC-mediated induction of unregulated cell growth involves posttranslational mechanisms that prevent proteasomal degradation of proto-oncogene β-catenin (CTNNB1) and its eventual translocation to the nucleus. However, about 10% of colorectal tumors also exhibit increased CTNNB1 mRNA. Here, we show in colorectal cancer that increased expression of ZNF148, the gene coding for transcription factor ZBP-89, correlated with reduced patient survival. Tissue arrays showed that ZBP-89 protein was overexpressed in the early stages of colorectal cancer. Conditional deletion of Zfp148 in a mouse model of Apc-mediated intestinal polyps demonstrated that ZBP-89 was required for polyp formation due to induction of Ctnnb1 gene expression. Chromatin immunoprecipitation (ChIP) and EMSA identified a ZBP-89–binding site in the proximal promoter of CTNNB1. Reciprocally, siRNA-mediated reduction of CTNNB1 expression also decreased ZBP-89 protein. ChIP identified TCF DNA binding sites in the ZNF148 promoter through which Wnt signaling regulates ZNF148 gene expression. Suppression of either ZNF148 or CTNNB1 reduced colony formation in WNT-dependent, but not WNT-independent cell lines. Therefore, the increase in intracellular β-catenin protein initiated by APC mutations is sustained by ZBP-89–mediated feedforward induction of CTNNB1 mRNA. Cancer Res; 76(23); 6877–87. ©2016 AACR.

BACKGROUND: The norepinephrine transporter (NET) is a high-affinity transporter for catecholamines. Its expression is almost exclusively restricted to the sympathetic nervous system. In this study we evaluated whether the NET can be used as a reporter gene for non-invasive imaging of genetically modified cells with radiolabeled probes. METHODS: Human A431, HT1080 and murine CMS-5 cells were retrovirally transduced with bovine NET cDNA. Transduced and parental cells were incubated in vitro with [(131)I]meta-iodobenzylguanidine ([(131)I]MIBG). The specificity of tracer uptake was determined by adding the NET inhibitor imipramine. Rat PC12 cells served as positive controls. Parental and A431NET cells were xenotransplanted into nude mice and tumor uptake of [(123)I]MIBG in vivo was determined after tracer administration. RESULTS: In vitro stably transduced cells showed a 66- to 120-fold higher [(131)I]MIBG uptake than parental cells. Incubation with imipramine reduced [(131)I]MIBG uptake of transduced cells to the level found in parental cells. More than 70% of the initial radioactivity was retained in all transduced cell lines after 2 h incubation with tracer-free medium. [(131)I]MIBG uptake in PC12 cells, which express the NET endogenously, was 20- to 28-fold lower than in transduced cells. In vivo, A431NET tumors demonstrated a 33-fold higher [(123)I]MIBG uptake than parental tumors. Gamma camera images 24 h after tracer injection showed no tracer uptake in parental A431 tumors, but clear images of A431NET tumors. CONCLUSIONS: Transduction of tumor cells with NET cDNA causes highly specific uptake and significant retention of catecholamine analogs in vitro and in vivo. These characteristics make the NET suitable as a reporter gene for non-invasive monitoring of gene transfer.

Tumour necrosis factor-α (TNFα) and polymorphonuclear neutrophils play key and interrelated roles in the inflammatory response against infectious agents. However, these entities can mediate significant tissue damage if their biological activity becomes deregulated. We have previously shown that canine hyperimmune frozen plasma (HFP) contains anti-TNFα activity that is attributable to elevated levels of soluble TNFα receptor 1 (sTNFR1). The aim of this study was to determine the effect of HFP on TNFα levels and neutrophil infiltration in a lipopolysaccharide (LPS)-mediated rat air pouch model of inflammation. Rats were administered either HFP, HFP which had been pre-incubated with anti-sTNFR1 antibody (5 ng/ml), fresh frozen plasma (FFP), physiological saline (PS) at 2 ml/day or Carprofen at 5 mg/kg for 3 days prior to LPS challenge. Pouch fluid was withdrawn at 1, 6, 12, 24 and 48 h post-LPS challenge and assayed for TNFα by ELISA, and for total leukocytes and neutrophils by microscopic examination. At 6 h post-LPS challenge, both TNFα levels and neutrophil counts were significantly lower in HFP-treated rats than was found in FFP, PS or Carprofen treated animals (p < 0.05). In a sTNFR1 blocking experiment, incubation of HFP with anti-sTNFR1 antibody resulted in significant increases in neutrophil numbers and TNFα levels, which suggests that the anti-TNFα activity observed in HFP may be due to elevated levels of sTNFR1. The data also revealed a significant inverse correlation between total leukocyte counts and sTNFR1 levels present in pouch fluid (r = −0.73, p < 0.0001). Our observations suggest that HFP warrants further investigation as a possible means for modulating acute inflammatory processes where TNFα is a key mediator.

Background & Aims ZBP-89 (also ZNF148 or Zfp148) is a butyrate-inducible zinc finger transcription factor that binds to GC-rich DNA elements. Deletion of the N-terminal domain is sufficient to increase mucosal susceptibility to chemical injury and inflammation. We investigated whether conditional deletion of ZBP-89 from the intestinal and colonic epithelium of mice increases their susceptibility to pathogens such as Salmonella typhimurium . Methods We generated mice with a conditional null allele of Zfp148 (ZBP-89 FL/FL ) using homologous recombination to flank Zfp148 with LoxP sites (ZBP-89 FL/FL ), and then bred the resulting mice with those that express VillinCre. We used microarray analysis to compare gene expression patterns in colonic mucosa between ZBP-89 ΔInt and C57BL/6 wild-type mice (controls). Mice were gavaged with 2 isogenic strains of S typhimurium after administration of streptomycin. Results Microarray analysis revealed that the colonic mucosa of ZBP-89 ΔInt mice had reduced levels of tryptophan hydroxylase 1 (Tph1) messenger RNA, encoding the rate-limiting enzyme in enterochromaffin cell serotonin (5-hydroxytryptamine [5HT]) biosynthesis. DNA affinity precipitation demonstrated direct binding of ZBP-89 to the mouse Tph1 promoter, which was required for its basal and butyrate-inducible expression. ZBP-89 ΔInt mice did not increase mucosal levels of 5HT in response to S typhimurium infection, and succumbed to the infection 2 days before control mice. The ΔhilA isogenic mutant of S typhimurium lacks this butyrate-regulated locus and stimulated, rather than suppressed, expression of Tph1 approximately 50-fold in control, but not ZBP-89 ΔInt , mice, correlating with fecal levels of butyrate. Conclusions ZBP-89 is required for butyrate-induced expression of the Tph1 gene and subsequent production of 5HT in response to bacterial infection in mice. Reductions in epithelial ZBP-89 increase susceptibility to colitis and sepsis after infection with S typhimurium , partly because of reduced induction of 5HT production in response to butyrate and decreased secretion of antimicrobial peptides.

The purpose of this study was to test the effect of canine hyperimmune frozen plasma (HFP), which is known to contain elevated levels of soluble TNFα receptor 1 (sTNFR1), on TNFα and inflammatory cell levels in a LPS-mediated rat air pouch model of inflammation. There appears to be a correlation between elevated levels of sTNFR1 and depression of TNFα and neutrophil levels in the pouch fluid of HFP dosed rats (r = -0.73, P < 0.0001). The data suggest that canine HFP, which has been demonstrated to contain elevated levels of sTNFR1 compared with FFP, has a direct effect on depressing TNFα levels and neutrophil sequestration in the rat air pouch model of inflammation. These data suggest that HFP may be worthy of further investigation to determine whether such preparations have a therapeutic potential for treatment of acute inflammatory diseases in which TNFα is implicated.