TTR exon-humanized mouse optimal for verifying new therapies for FAP.

Familial amyloidotic polyneuropathy (FAP) is caused by a mutation in the transthyretin (TTR) gene. In addition, deposition of wild-type TTR can cause senile systemic amyloidosis (SSA). To date, we have produced several transgenic mouse models for FAP and SSA by introducing TTR genes with different promoters or mutations. However, mouse TTR can associate with human TTR to produce hybrid tetramers in transgenic mice. Thus, these transgenic mice cannot be used to test the efficacy of a new therapy. In this study, we attempted to construct an optimized mouse model to verify a new therapy. The TTR gene consists of 4 exons and 3 introns. We prepared two gRNAs, one for the exon 1 and the other for exon 4, and a single donor vector carrying the whole TTR gene in which mouse exons were replaced with human exons. Using these vectors, we produced a TTR exon-humanized mouse with human exons and mouse introns using genome editing technology. These TTR exon-humanized mice showed normal TTR expression patterns in terms of serum TTR level and spatial specificity. These TTR exon-humanized mice will be useful for devising new treatment methods for FAP, including gene therapy.

Transgenic mouse model for imaging of ATF4 translational activation-related cellular stress responses in vivo.

Activating transcription factor 4 (ATF4) is a translationally activated protein that plays a role in cellular adaptation to several stresses. Because these stresses are associated with various diseases, the translational control of ATF4 needs to be evaluated from the physiological and pathological points of view. We have developed a transgenic mouse model to monitor the translational activation of ATF4 in response to cellular stress. By using this mouse model, we were able to detect nutrient starvation response, antivirus response, endoplasmic reticulum (ER) stress response, and oxidative stress in vitro and ex vivo, as well as in vivo. The reporter system introduced into our mouse model was also shown to work in a stress intensity-dependent manner and a stress duration-dependent manner. The mouse model is therefore a useful tool for imaging ATF4 translational activation at various levels, from cell cultures to whole bodies, and it has a range of useful applications in investigations on the physiological and pathological roles of ATF4-related stress and in the development of clinical drugs for treating ATF4-associated diseases.

Transgenic mouse model for imaging of interleukin-1ß-related inflammation in vivo.

Inflammation is a biological response associated with symptoms of various diseases, and its study is important in gaining an understanding of the pathological conditions of such diseases and in making strategic plans for promoting healing. It is therefore essential to develop technologies for the detection of inflammatory conditions. Interleukin-1ß (IL-1ß) is a proinflammatory cytokine produced and secreted mainly by monocytes and macrophages in response to inflammatory stimulation. The activation of IL-1ß is regulated through transcriptional induction by the promoter and post-translational processing by the inflammasome. Here we have developed a reporter gene to monitor the activation status of IL-1ß by using a dual regulation system and, by using the reporter gene, we have established a mouse model that permits low-invasive visualization of the inflammatory status. Previous reporter systems dependent on the transcription or processing of IL-1ß show problems in terms of background noise or signal specificity. Our reporter system overcomes these weaknesses by combining advantages from regulation by a promoter and processing of IL-1ß. Our mouse model detected specific physiological inflammation in the liver and pancreas caused by hepatitis or pancreatitis models, respectively. Our reporter gene and mouse model are therefore expected to become useful bioresources for future medical science.

A reporter mouse for optical imaging of inflammation in mdx muscles.

BACKGROUND: Duchenne muscular dystrophy (DMD) is due to mutations in the gene coding for human DMD; DMD is characterized by progressive muscle degeneration, inflammation, fat accumulation, and fibrosis. The mdx mouse model of DMD lacks dystrophin protein and undergoes a predictable disease course. While this model has been a valuable resource for pre-clinical studies aiming to test therapeutic compounds, its utility is compromised by a lack of reliable biochemical tools to quantifiably assay muscle disease. Additionally, there are few non-invasive assays available to researchers for measuring early indicators of disease progression in mdx mice. METHODS: Mdx mice were crossed to knock-in mice expressing luciferase from the Cox2 promoter. These reporter mice (Cox2 (FLuc/+) DMD (-/-) ) were created to serve as a tool for researchers to evaluate muscle inflammation. Luciferase expression was assayed by immunohistochemistry to insure that it correlated with muscle lesions. The luciferase signal was quantified by optical imaging and luciferase assays to verify that the signal correlated with muscle damage. As proof of principle, Cox2 (FLuc/+) DMD (-/-) mice were also treated with prednisolone to validate that a reduction in luciferase signal correlated with prednisone treatment. RESULTS: In this investigation, a novel reporter mouse (Cox2 (FLuc/+) DMD (-/-) mice) was created and validated for non-invasive quantification of muscle inflammation in vivo. In this dystrophic mouse, luciferase is expressed from cyclooxygenase 2 (Cox2) expressing cells and bioluminescence is detected by optical imaging. Bioluminescence is significantly enhanced in damaged muscle of exercised Cox2 (FLuc/+) DMD (-/-) mice compared to non-exercised Cox2 (FLuc/+) DMD (+/+) mice. Moreover, the Cox2 bioluminescent signal is reduced in Cox2 (FLuc/+) DMD (-/-) mice in response to a course of steroid treatment. Reduction in bioluminescence is detectable prior to measurable therapy-elicited improvements in muscle strength, as assessed by traditional means. Biochemical assay of luciferase provides a second means to quantify muscle inflammation. CONCLUSIONS: The Cox2 (FLuc/+) DMD (-/-) mouse is a novel tool to evaluate the therapeutic benefits of drugs intended to target inflammatory aspects of dystrophic pathology. This mouse model will be a useful adjunct to traditional outcome measures in assessing potential therapeutic compounds.

Targeted deletion and lipidomic analysis identify epithelial cell COX-2 as a major driver of chemically induced skin cancer.

UNLABELLED: Pharmacologic and global gene deletion studies demonstrate that cyclooxygenase-2 (PTGS2/COX-2) plays a critical role in DMBA/TPA-induced skin tumor induction. Although many cell types in the tumor microenvironment express COX-2, the cell types in which COX-2 expression is required for tumor promotion are not clearly established. Here, cell type-specific Cox-2 gene deletion reveals a vital role for skin epithelial cell COX-2 expression in DMBA/TPA tumor induction. In contrast, myeloid Cox-2 gene deletion has no effect on DMBA/TPA tumorigenesis. The infrequent, small tumors that develop on mice with an epithelial cell-specific Cox-2 gene deletion have decreased proliferation and increased cell differentiation properties. Blood vessel density is reduced in tumors with an epithelial cell-specific Cox-2 gene deletion, compared with littermate control tumors, suggesting a reciprocal relationship in tumor progression between COX-2-expressing tumor epithelial cells and microenvironment endothelial cells. Lipidomics analysis of skin and tumors from DMBA/TPA-treated mice suggests that the prostaglandins PGE2 and PGF2α are likely candidates for the epithelial cell COX-2-dependent eicosanoids that mediate tumor progression. This study both illustrates the value of cell type-specific gene deletions in understanding the cellular roles of signal-generating pathways in complex microenvironments and emphasizes the benefit of a systems-based lipidomic analysis approach to identify candidate lipid mediators of biologic responses. IMPLICATIONS: Cox-2 gene deletion demonstrates that intrinsic COX-2 expression in initiated keratinocytes is a principal driver of skin carcinogenesis; lipidomic analysis identifies likely prostanoid effectors.

Reversible suppression of cyclooxygenase 2 (COX-2) expression in vivo by inducible RNA interference.

Prostaglandin-endoperoxide synthase 2 (PTGS2), also known as cyclooxygenase 2 (COX-2), plays a critical role in many normal physiological functions and modulates a variety of pathological conditions. The ability to turn endogenous COX-2 on and off in a reversible fashion, at specific times and in specific cell types, would be a powerful tool in determining its role in many contexts. To achieve this goal, we took advantage of a recently developed RNA interference system in mice. An shRNA targeting the Cox2 mRNA 3'untranslated region was inserted into a microRNA expression cassette, under the control of a tetracycline response element (TRE) promoter. Transgenic mice containing the COX-2-shRNA were crossed with mice encoding a CAG promoter-driven reverse tetracycline transactivator, which activates the TRE promoter in the presence of tetracycline/doxycycline. To facilitate testing the system, we generated a knockin reporter mouse in which the firefly luciferase gene replaces the Cox2 coding region. Cox2 promoter activation in cultured cells from triple transgenic mice containing the luciferase allele, the shRNA and the transactivator transgene resulted in robust luciferase and COX-2 expression that was reversibly down-regulated by doxycycline administration. In vivo, using a skin inflammation-model, both luciferase and COX-2 expression were inhibited over 80% in mice that received doxycycline in their diet, leading to a significant reduction of infiltrating leukocytes. In summary, using inducible RNA interference to target COX-2 expression, we demonstrate potent, reversible Cox2 gene silencing in vivo. This system should provide a valuable tool to analyze cell type-specific roles for COX-2.

Hepatic ischemia and reperfusion injury in the absence of myeloid cell-derived COX-2 in mice.

Cyclooxygenase-2 (COX-2) is a mediator of hepatic ischemia and reperfusion injury (IRI). While both global COX-2 deletion and pharmacologic COX-2 inhibition ameliorate liver IRI, the clinical use of COX-2 inhibitors has been linked to increased risks of heart attack and stroke. Therefore, a better understanding of the role of COX-2 in different cell types may lead to improved therapeutic strategies for hepatic IRI. Macrophages of myeloid origin are currently considered to be important sources of the COX-2 in damaged livers. Here, we used a Cox-2flox conditional knockout mouse (COX-2-M/-M) to examine the function of COX-2 expression in myeloid cells during liver IRI. COX-2-M/-M mice and their WT control littermates were subjected to partial liver ischemia followed by reperfusion. COX-2-M/-M macrophages did not express COX-2 upon lipopolysaccharide stimulation and COX-2-M/-M livers showed reduced levels of COX-2 protein post-IRI. Nevertheless, selective deletion of myeloid cell-derived COX-2 failed to ameliorate liver IRI; serum transaminases and histology were comparable in both COX-2-M/-M and WT mice. COX-2-M/-M livers, like WT livers, developed extensive necrosis, vascular congestion, leukocyte infiltration and matrix metalloproteinase-9 (MMP-9) expression post-reperfusion. In addition, myeloid COX-2 deletion led to a transient increase in IL-6 levels after hepatic reperfusion, when compared to controls. Administration of celecoxib, a selective COX-2 inhibitor, resulted in significantly improved liver function and histology in both COX-2-M/-M and WT mice post-reperfusion, providing evidence that COX-2-mediated liver IRI is caused by COX-2 derived from a source(s) other than myeloid cells. In conclusion, these results support the view that myeloid COX-2, including myeloid-macrophage COX-2, is not responsible for the hepatic IRI phenotype.

Candidate chromosome 1 disease susceptibility genes for Sjogren's syndrome xerostomia are narrowed by novel NOD.B10 congenic mice.

Sjogren's syndrome (SS) is characterized by salivary gland leukocytic infiltrates and impaired salivation (xerostomia). Cox-2 (Ptgs2) is located on chromosome 1 within the span of the Aec2 region. In an attempt to demonstrate that COX-2 drives antibody-dependent hyposalivation, NOD.B10 congenic mice bearing a Cox-2flox gene were generated. A congenic line with non-NOD alleles in Cox-2-flanking genes failed manifest xerostomia. Further backcrossing yielded disease-susceptible NOD.B10 Cox-2flox lines; fine genetic mapping determined that critical Aec2 genes lie within a 1.56 to 2.17Mb span of DNA downstream of Cox-2. Bioinformatics analysis revealed that susceptible and non-susceptible lines exhibit non-synonymous coding SNPs in 8 protein-encoding genes of this region, thereby better delineating candidate Aec2 alleles needed for SS xerostomia.

Cell-type-specific roles for COX-2 in UVB-induced skin cancer.

In human tumors, and in mouse models, cyclooxygenase-2 (COX-2) levels are frequently correlated with tumor development/burden. In addition to intrinsic tumor cell expression, COX-2 is often present in fibroblasts, myofibroblasts and endothelial cells of the tumor microenvironment, and in infiltrating immune cells. Intrinsic cancer cell COX-2 expression is postulated as only one of many sources for prostanoids required for tumor promotion/progression. Although both COX-2 inhibition and global Cox-2 gene deletion ameliorate ultraviolet B (UVB)-induced SKH-1 mouse skin tumorigenesis, neither manipulation can elucidate the cell type(s) in which COX-2 expression is required for tumorigenesis; both eliminate COX-2 activity in all cells. To address this question, we created Cox-2(flox/flox) mice, in which the Cox-2 gene can be eliminated in a cell-type-specific fashion by targeted Cre recombinase expression. Cox-2 deletion in skin epithelial cells of SKH-1 Cox-2(flox/flox);K14Cre(+) mice resulted, following UVB irradiation, in reduced skin hyperplasia and increased apoptosis. Targeted epithelial cell Cox-2 deletion also resulted in reduced tumor incidence, frequency, size and proliferation rate, altered tumor cell differentiation and reduced tumor vascularization. Moreover, Cox-2(flox/flox);K14Cre(+) papillomas did not progress to squamous cell carcinomas. In contrast, Cox-2 deletion in SKH-1 Cox-2(flox/flox); LysMCre(+) myeloid cells had no effect on UVB tumor induction. We conclude that (i) intrinsic epithelial COX-2 activity plays a major role in UVB-induced skin cancer, (ii) macrophage/myeloid COX-2 plays no role in UVB-induced skin cancer and (iii) either there may be another COX-2-dependent prostanoid source(s) that drives UVB skin tumor induction or there may exist a COX-2-independent pathway(s) to UVB-induced skin cancer.

Context-dependent activation of Wnt signaling by tumor suppressor RUNX3 in gastric cancer cells.

RUNX3 is a tumor suppressor for a variety of cancers. RUNX3 suppresses the canonical Wnt signaling pathway by binding to the TCF4/ß-catenin complex, resulting in the inhibition of binding of the complex to the Wnt target gene promoter. Here, we confirmed that RUNX3 suppressed Wnt signaling activity in several gastric cancer cell lines; however, we found that RUNX3 increased the Wnt signaling activity in KatoIII and SNU668 gastric cancer cells. Notably, RUNX3 expression increased the ratio of the Wnt signaling-high population in the KatoIII cells. although the maximum Wnt activation level of individual cells was similar to that in the control. As found previously, RUNX3 also binds to TCF4 and ß-catenin in KatoIII cells, suggesting that these molecules form a ternary complex. Moreover, the ChIP analyses revealed that TCF4, ß-catenin and RUNX3 bind the promoter region of the Wnt target genes, Axin2 and c-Myc, and the occupancy of TCF4 and ß-catenin in these promoter regions is increased by the RUNX3 expression. These results suggest that RUNX3 stabilizes the TCF4/ß-catenin complex on the Wnt target gene promoter in KatoIII cells, leading to activation of Wnt signaling. Although RUNX3 increased the Wnt signaling activity, its expression resulted in suppression of tumorigenesis of KatoIII cells, indicating that RUNX3 plays a tumor-suppressing role in KatoIII cells through a Wnt-independent mechanism. These results indicate that RUNX3 can either suppress or activate the Wnt signaling pathway through its binding to the TCF4/ß-catenin complex by cell context-dependent mechanisms.

Targeted Cox2 gene deletion in intestinal epithelial cells decreases tumorigenesis in female, but not male, ApcMin/+ mice.

Mice heterozygous for mutations in the adenomatous polyposis coli gene (Apc(+/-) mice) develop intestinal neoplasia. Apc(+/-) tumor formation is thought to be dependent on cyclooxygenase 2 (COX2) expression; both pharmacologic COX2 inhibition and global Cox2 gene deletion reduce the number of intestinal tumors in Apc(+/-) mice. COX2 expression is reported in epithelial cells, fibroblasts, macrophages and endothelial cells of Apc(+/-) mouse polyps. However, the cell type(s) in which COX2 expression is required for Apc(+/-) tumor induction is not known. To address this question, we developed Apc(Min/+) mice in which the Cox2 gene is specifically deleted either in intestinal epithelial cells or in myeloid cells. There is no significant difference in intestinal polyp number between Apc(Min/+) mice with a targeted Cox2 gene deletion in myeloid cells and their control littermate Apc(Min/+) mice. In contrast, Apc(Min/+) mice with a targeted Cox2 deletion in intestinal epithelial cells have reduced intestinal tumorigenesis when compared to their littermate control Apc(Min/+) mice. However, two gender-specific effects are notable. First, female Apc(Min/+) mice developed more intestinal tumors than male Apc(Min/+) mice. Second, targeted intestinal epithelial cell Cox2 deletion decreased tumorigenesis in female, but not in male, Apc(Min/+) mice. Considered in the light of pharmacologic studies and studies with global Cox2 gene knockout mice, our data suggest that (i) intrinsic COX2 expression in intestinal epithelial cells plays a gender-specific role in tumor development in Apc(Min/+) mice, and (ii) COX2 expression in cell type(s) other than intestinal epithelial cells also modulates intestinal tumorigenesis in Apc(Min/+) mice, by a paracrine process.

STAT3-driven upregulation of TLR2 promotes gastric tumorigenesis independent of tumor inflammation.

Gastric cancer (GC) is associated with chronic inflammation; however, the molecular mechanisms promoting tumorigenesis remain ill defined. Using a GC mouse model driven by hyperactivation of the signal transducer and activator of transcription (STAT)3 oncogene, we show that STAT3 directly upregulates the epithelial expression of the inflammatory mediator Toll-like receptor (TLR)2 in gastric tumors. Genetic and therapeutic targeting of TLR2 inhibited gastric tumorigenesis, but not inflammation, characterized by reduced proliferation and increased apoptosis of the gastric epithelium. Increased STAT3 pathway activation and TLR2 expression were also associated with poor GC patient survival. Collectively, our data reveal an unexpected role for TLR2 in the oncogenic function of STAT3 that may represent a therapeutic target in GC.

Activation of epidermal growth factor receptor signaling by the prostaglandin E(2) receptor EP4 pathway during gastric tumorigenesis.

Cyclooxygenase-2 (COX-2) plays an important role in tumorigenesis through prostaglandin E(2) (PGE(2)) biosynthesis. It has been shown by in vitro studies that PGE(2) signaling transactivates epidermal growth factor receptor (EGFR) through an intracellular mechanism. However, the mechanisms underlying PGE(2)-induced EGFR activation in in vivo tumors are still not fully understood. We previously constructed transgenic mice that develop gastric tumors caused by oncogenic activation and PGE(2) pathway induction. Importantly, expression of EGFR ligands, epiregulin, amphiregulin, heparin-binding EGF-like growth factor, and betacellulin, as well as a disintegrin and metalloproteinases (ADAMs), ADAM8, ADAM9, ADAM10, and ADAM17 were significantly increased in the mouse gastric tumors in a PGE(2) pathway-dependent manner. These ADAMs can activate EGFR by ectodomain shedding of EGFR ligands. Notably, the extensive induction of EGFR ligands and ADAMs was suppressed by inhibition of the PGE(2) receptor EP4. Moreover, EP4 signaling induced expression of amphiregulin and epiregulin in activated macrophages, whereas EP4 pathway was required for basal expression of epiregulin in gastric epithelial cells. In contrast, ADAMs were not induced directly by PGE(2) in these cells, suggesting indirect mechanism possibly through PGE(2)-associated inflammatory responses. These results suggest that PGE(2) signaling through EP4 activates EGFR in gastric tumors through global induction of EGFR ligands and ADAMs in several cell types either by direct or indirect mechanism. Importantly, gastric tumorigenesis of the transgenic mice was significantly suppressed by combination treatment with EGFR and COX-2 inhibitors. Therefore, it is possible that inhibition of both COX-2/PGE(2) and EGFR pathways represents an effective strategy for preventing gastric cancer.

Conditional bicistronic Cre reporter line expressing both firefly luciferase and ß-galactosidase.

PURPOSE: The Cre-loxP system has become an important strategy for conditional gene deletion and conditional gene expression in genetically engineered mice. To evaluate Cre recombinase expression, we generated reporter mice that permit both noninvasive imaging in living animals and either ex vivo histochemical/immunohistochemical tissue transgene expression analysis or quantitative enzyme analysis in the same animal. PROCEDURES: Transgenic reporter mice were generated in which a loxP-flanked enhanced green fluorescent protein (EGFP) reporter gene and STOP sequence are placed after the nearly ubiquitously expressed CAG promoter, but before a bicistronic transcriptional unit containing luciferase and ß-galactosidase reporter gene coding sequences. RESULTS: After global deletion of the floxed STOP sequence by germ line Cre deletion, the reporter mouse expresses luciferase and ß-galactosidase in all tissues examined. Tissue-specific expression of both reporter genes occurs in reporter mouse strains expressing Cre in skin (K14 keratin Cre), heart (myosin light chair Cre), or colon (Villin Cre). CONCLUSION: The luc-gal(Tg) reporter mouse allows noninvasive imaging of target Cre activation both in living animals and in tissues and cells following necropsy, using loss of EGFP expression, gain of luciferase expression, and gain of ß-galactosidase expression as alternatives within the same animal for qualitative analysis of Cre expression.

Cox-2 deletion in myeloid and endothelial cells, but not in epithelial cells, exacerbates murine colitis.

Patients with inflammatory bowel diseases are at increased risk for colorectal cancer. Pharmacological inhibition of cyclooxygenase (COX) function exacerbates symptoms in colitis patients. Animal models of colitis using Cox-2-knockout mice and COX inhibitors also indicate that COX-2 has a protective role against colon inflammation. However, because conventional Cox-2 deletion and COX-2 inhibitors eliminate COX-2 function in all cells, it has not been possible to analyze the role(s) of COX-2 in different cell types. Here, we use a Cox-2(flox) conditional knockout mouse to analyze the role of COX-2 expression in distinct cell types in the colon in response to dextran sulfate sodium (DSS)-induced colitis. We generated Cox-2 conditional knockouts in myeloid cells with LysMCre knock-in mice, in endothelial cells with VECadCreERT2 transgenic mice and in epithelial cells with VillinCre transgenic mice. When treated with DSS to induce colitis, both myeloid cell-specific and endothelial cell-specific Cox-2-knockout mice exhibited greater weight loss, increased clinical scores and decreased epithelial cell proliferation after DSS injury when compared with littermate controls. In contrast, epithelial-specific Cox-2 knockouts and control littermates did not differ in response to DSS. These results suggest that COX-2 expression in myeloid cells and endothelial cells, but not epithelial cells, is important for protection of epithelial cells in this murine colitis model.

Prostaglandin E2 signaling and bacterial infection recruit tumor-promoting macrophages to mouse gastric tumors.

BACKGROUND & AIMS: Helicobacter pylori infection induces an inflammatory response, which can contribute to gastric tumorigenesis. Induction of cyclooxygenase-2 (COX-2) results in production of prostaglandin E(2) (PGE(2)), which mediates inflammation. We investigated the roles of bacterial infection and PGE(2) signaling in gastric tumorigenesis in mice. METHODS: We generated a germfree (GF) colony of K19-Wnt1/C2mE mice (Gan mice); these mice develop gastric cancer. We examined tumor phenotypes, expression of cytokines and chemokines, and recruitment of macrophages. We also investigated PGE(2) signaling through the PGE(2) receptor subtype 4 (EP4) in Gan mice given specific inhibitors. RESULTS: Gan mice raised in a specific pathogen-free facility developed large gastric tumors, whereas gastric tumorigenesis was significantly suppressed in GF-Gan mice; reconstitution of commensal flora or infection with Helicobacter felis induced gastric tumor development in these mice. Macrophage infiltration was significantly suppressed in the stomachs of GF-Gan mice. Gan mice given an EP4 inhibitor had decreased expression of cytokines and chemokines. PGE(2) signaling and bacterial infection or stimulation with lipopolysaccharide induced expression of the chemokine C-C motif ligand 2 (CCL2) (which attracts macrophage) in tumor stromal cells or cultured macrophages, respectively. CCL2 inhibition suppressed macrophage infiltration in tumors, and depletion of macrophages from the tumors of Gan mice led to signs of tumor regression. Wnt signaling was suppressed in the tumors of GF-Gan and Gan mice given injections of tumor necrosis factor-α neutralizing antibody. CONCLUSIONS: Bacterial infection and PGE(2) signaling are required for gastric tumorigenesis in mice; they cooperate to up-regulate CCL2, which recruits macrophage to gastric tumors. Macrophage-derived tumor necrosis factor-α promotes Wnt signaling in epithelial cells, which contributes to gastric tumorigenesis.

Increased lysis of stem cells but not their differentiated cells by natural killer cells; de-differentiation or reprogramming activates NK cells.

The aims of this study are to demonstrate the increased lysis of stem cells but not their differentiated counterparts by the NK cells and to determine whether disturbance in cell differentiation is a cause for increased sensitivity to NK cell mediated cytotoxicity. Increased cytotoxicity and augmented secretion of IFN-gamma were both observed when PBMCs or NK cells were co-incubated with primary UCLA oral squamous carcinoma stem cells (UCLA-OSCSCs) when compared to differentiated UCLA oral squamous carcinoma cells (UCLA-OSCCs). In addition, human embryonic stem cells (hESCs) were also lysed greatly by the NK cells. Moreover, NK cells were found to lyse human Mesenchymal Stem Cells (hMSCs), human dental pulp stem cells (hDPSCs) and human induced pluripotent stem cells (hiPSCs) significantly more than their differentiated counterparts or parental lines from which they were derived. It was also found that inhibition of differentiation or reversion of cells to a less-differentiated phenotype by blocking NFkappaB or targeted knock down of COX2 in monocytes significantly augmented NK cell cytotoxicity and secretion of IFN-gamma. Taken together, these results suggest that stem cells are significant targets of the NK cell cytotoxicity. However, to support differentiation of a subset of tumor or healthy untransformed primary stem cells, NK cells may be required to lyse a number of stem cells and/or those which are either defective or incapable of full differentiation in order to lose their cytotoxic function and gain the ability to secrete cytokines (split anergy). Therefore, patients with cancer may benefit from repeated allogeneic NK cell transplantation for specific elimination of cancer stem cells.

Cox-2 gene expression in chemically induced skin papillomas cannot predict subsequent tumor fate.

Elevated cyclooxygenase-2 (COX-2) expression is observed in a variety of premalignant neoplastic tissues, suggesting COX-2 expression might serve as a potential indicator of subsequent tumor development. However, it has not been possible to compare the relationship between Cox-2 gene expression in premalignant lesions and their subsequent fate, because conventional studies require tissue destruction for analysis of gene expression. To monitor COX-2 expression non-invasively during tumor development, we created a Cox-2 luciferase knock-in mouse, Cox-2(luc), in which the firefly luciferase coding region replaces the Cox-2 coding region. Luciferase activity was non-invasively, quantitatively and repeatedly monitored in Cox-2(luc/+) mice subjected to DMBA/TPA multistage skin tumor induction. Luciferase activity is significantly higher in all papillomas than in surrounding skin. However, the magnitude of Cox-2 promoter-driven luciferase activity in small papillomas cannot predict subsequent papilloma regression or growth. Elevated Cox-2 promoter-driven luciferase signal can be detected when papillomas first become visible, but not before this time.

Preserved heart function and maintained response to cardiac stresses in a genetic model of cardiomyocyte-targeted deficiency of cyclooxygenase-2.

Cyclooxygenase-1 and -2 are rate-limiting enzymes in the formation of a wide array of bioactive lipid mediators collectively known as prostanoids (prostaglandins, prostacyclins, and thromboxanes). Evidence from clinical trials shows that selective inhibition of the second isoenzyme (cyclooxygenase-2, or Cox-2) is associated with increased risk for serious cardiovascular events and findings from animal-based studies have suggested protective roles of Cox-2 for the heart. To further characterize the function of Cox-2 in the heart, mice with loxP sites flanking exons 4 and 5 of Cox-2 were rendered knockout specifically in cardiac myocytes (Cox-2 CKO mice) via cre-mediated recombination. Baseline cardiac performance of CKO mice remained unchanged and closely resembled that of control mice. Furthermore, myocardial infarct size induced after in vivo ischemia/reperfusion (I/R) injury was comparable between CKO and control mice. In addition, cardiac hypertrophy and function four weeks after transverse aortic constriction (TAC) was found to be similar between the two groups. Assessment of Cox-2 expression in purified adult cardiac cells isolated after I/R and TAC suggests that the dominant source of Cox-2 is found in the non-myocyte fraction. In conclusion, our animal-based analyses together with the cell-based observations portray a limited role of cardiomyocyte-produced Cox-2 at baseline and in the context of ischemic or hemodynamic challenge.

Novel anti-inflammatory functions for endothelial and myeloid cyclooxygenase-2 in a new mouse model of Crohn's disease.

Cyclooxygenase-2 (COX-2) is an important regulator of inflammation implicated in the development of a variety of diseases, including inflammatory bowel disease (IBD). However, the regulation of intestinal inflammation by COX-2 is poorly understood. We previously reported that COX-2(-/-) mice fed a cholate-containing high-fat (CCHF) diet had high mortality of unknown mechanisms attributable to severe intestinal inflammation in the ileo-ceco-colic junction that presented characteristics similar to Crohn's disease (CD). To further characterize the role of COX-2 in intestinal inflammation, we established cell-specific conditional COX-2(-/-) mice. Endothelial cell-specific (COX-2(-E/-E)) and myeloid cell-specific (COX-2(-M/-M)) COX-2(-/-) mice, but not wild-type mice, on the CCHF diet developed localized CD-like pathology at the ileo-ceco-colic junction that was associated with cellular infiltration, increased expression of myeloperoxidase and IL-5, and decreased IL-10 expression. The CD-like pathology in COX-2(-E/-E) mice was also accompanied by increased expression of cytokines (IL-6, TNF-alpha, and INF-gamma), compared with wild-type mice and COX-2(-M/-M) mice. In contrast, the ileo-ceco-colic inflammation in COX-2(-M/-M) mice was associated with more pronounced infiltration of granulocytes and macrophages than COX-2(-E/-E) mice. COX-2(-ME/-ME) (COX-2(-M/-M) x COX-2(-E/-E)) mice on the CCHF diet developed CD-like pathology in the ileo-ceco-colic junction reminiscent of total COX-2(-/-) mice on CCHF diet and wild-type mice on CCHF diet treated with COX-2 inhibitor, celecoxib. The pathology of diet-mediated ileo-ceco-colic inflammation in COX-2(-/-) mice offers an excellent model system to elucidate the protective roles of endothelial and myeloid COX-2 and the molecular pathogenesis of CD.