Development Of Transgenic Mice Research Articles

Abstract 603: Effective And Sustained ACE2 Delivery Using Minicircles Technology

Chronic and sustained amplification of ACE2 activity in vivo has required the development of transgenic mice or the use of viral vectors. Minicircle is a new gene delivery technology which is resistant to gene silencing, and therefore represents an attractive platform for gene replacement strategies in vivo . Here we cloned cDNA of soluble mouse ACE2 into a circular expression cassette and the resulting ACE2 minicircle (MC) was injected to female FVB mice using iv. hydrodynamic approach (10ug or 30ug/mouse). At 3-7d after MC administration, serum ACE2 activity in mice that received 10ug ACE2MC (n=9) was over 100-fold higher than in controls (n=9) (138±48 vs 0.7±0.2 RFU/uL/hr) and in ACE2MC mice (30ug) (n=8) was almost 1000-fold higher than in controls (n=14) (480 ±153 vs 0.5±0.1 RFU/uL/hr, respectively). Mice that received 10 ug ACE2MC were followed for consecutive serum ACE2 activity monitoring, BP measurements and plasma Ang levels. The increase in serum ACE2 activity was sustained until the end of the study (up to 82 days) (Figure). Despite such a marked increase in serum ACE2 activity in ACE2MC mice, conscious SBP was not different from controls (137±8 vs 138±7 mmHg, respectively). At the end of the study, when Ang II was infused acutely (0.2 ug/kg BW i.p.), the increase in plasma Ang II in ACE2MC mice was significantly reduced compared to control mice (915±154 vs 1420±131 fmoL/mL, p<0.05). Mini-circle delivery of ACE2 results in a dose-dependent and sustained long-term increase in serum ACE2 that efficiently degrades plasma Ang II. Extremely high increases in serum ACE2 activity do not reduce BP probably due to activation of non-ACE2 dependent compensatory Ang-hydrolyzing pathways.

Abstract 100A: Prognostic significance of the expression levels of target genes of retinoic acid in hepatocellular carcinoma.

Abstract Hepatocellular carcinoma (HCC), a common and fatal malignancy, is increasing in incidence in developed countries. HCC generally arises in cirrhotic livers and has a poor prognosis because of the high incidence of disease recurrence after curative treatment. To improve patient prognosis, identification of new targets for early detection, chemoprevention, and treatment is urgently required. In the pathogenesis of chronic liver disease, activation of hepatic stellate cells accompanied by decreasing vitamin A storage has been observed, leading to development of the hypothesis that the loss of retinoid content is closely associated with the progression of chronic liver diseases including HCC. In a previous study, we observed steatohepatitis and HCC development in transgenic mice that express a dominant-negative form of RAR in a liver-specific manner. These findings indicate that identifying genes regulated by retinoid signaling is important for understanding the molecular mechanisms of liver disease leading to hepatocarcinogenesis. To identify novel retinoic acid (RA)-responsive genes facilitating the antitumor activity of RA in hepatocellular carcinoma (HCC), we performed a gene screening based on in silico analysis of retinoic acid response elements (RAREs), chromatin immunoprecipitation analysis of the retinoic acid receptor (RAR), and gene expression analysis. We identified 201 candidate genes with promoter regions containing consensus RAREs using in silico analysis. We identified 126 candidate genes with functional RAREs which are occupied by RAR and finally selected 26 RA-responsive genes in 2 human HCC and 1 human breast cancer cell lines. Among these genes, we identified OTU domain-containing 7B (OTUD7B) and frizzled family receptor 4 (FZD4) as potential inducers of RA-mediated antitumor activity. OTUD7B and FZD4 were involved in the regulation of nuclear factor κB and Wnt/β-catenin signaling, respectively, and affected the viability of HCC cell lines. To evaluate the clinical significance of RA-responsive genes in HCC, gene expression analysis was performed using tumor and nontumor tissues from 171 patients. In univariate and multivariate analyses, low OTUD7B mRNA expression in tumor tissue was associated with a poor prognosis forHCC patients. Conclusion: We identified RA-responsive genes that exhibit antitumor activity against HCC cells. These findings provide useful information to understand the molecular mechanisms underlying the chemopreventive effects of RA against liver diseases, including HCC. Citation Format: Goshi Shiota, Keita Kanki. Prognostic significance of the expression levels of target genes of retinoic acid in hepatocellular carcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 100A. doi:10.1158/1538-7445.AM2013-100A

Zbtb20 Defines a Hippocampal Neuronal Identity Through Direct Repression of Genes That Control Projection Neuron Development in the Isocortex

Hippocampal pyramidal neurons are important for encoding and retrieval of spatial maps and episodic memories. While previous work has shown that Zbtb20 is a cell fate determinant for CA1 pyramidal neurons, the regulatory mechanisms governing this process are not known. In this study, we demonstrate that Zbtb20 binds to genes that control neuronal subtype specification in the developing isocortex, including Cux1, Cux2, Fezf2, Foxp2, Mef2c, Rorb, Satb2, Sox5, Tbr1, Tle4, and Zfpm2. We show that Zbtb20 represses these genes during ectopic CA1 pyramidal neuron development in transgenic mice. These data reveal a novel regulatory mechanism by which Zbtb20 suppresses the acquisition of an isocortical fate during archicortical neurogenesis to ensure commitment to a CA1 pyramidal neuron fate. We further show that the expression pattern of Zbtb20 is evolutionary conserved in the fetal human hippocampus, where it is complementary to the expression pattern of the Zbtb20 target gene Tbr1. Therefore, the disclosed Zbtb20-mediated transcriptional repressor mechanism may be involved in development of the human archicortex.

Development of Transgenic Mice Containing an Introduced Stop Codon on the Human Methylmalonyl-CoA Mutase Locus

The mutation R403stop was found in an individual with mut0 methylmalonic aciduria (MMA) which resulted from a single base change of C→T in exon 6 of the methylmalonyl-CoA mutase gene (producing a TGA stop codon). In order to accurately model the human MMA disorder we introduced this mutation onto the human methylmalonyl-CoA mutase locus of a bacterial artificial chromosome. A mouse model was developed using this construct.The transgene was found to be intact in the mouse model, with 7 copies integrated at a single site in chromosome 3. The phenotype of the hemizygous mouse was unchanged until crossed against a methylmalonyl-CoA mutase knockout mouse. Pups with no endogenous mouse methylmalonyl-CoA mutase and one copy of the transgene became ill and died within 24 hours. This severe phenotype could be partially rescued by the addition of a transgene carrying two copies of the normal human methylmalonyl-CoA mutase locus. The “humanized” mice were smaller than control litter mates and had high levels of methylmalonic acid in their blood and tissues.This new transgenic MMA stop codon model mimics (at both the phenotypic and genotypic levels) the key features of the human MMA disorder. It will allow the trialing of pharmacological and, cell and gene therapies for the treatment of MMA and other human metabolic disorders caused by stop codon mutations.

Involvement of Host Stroma Cells and Tissue Fibrosis in Pancreatic Tumor Development in Transgenic Mice

IntroductionStroma cells and extracellular matrix (ECM) components provide the pivotal microenvironment for tumor development. The study aimed to evaluate the importance of the pancreatic stroma for tumor development.MethodsPancreatic tumor cells were implanted subcutaneously into green fluorescent protein transgenic mice, and stroma cells invading the tumors were identified through immunohistochemistry. Inhibition of tumor invasion by stroma cells was achieved with halofuginone, an inhibitor of TGFβ/Smad3 signaling, alone or in combination with chemotherapy. The origin of tumor ECM was evaluated with species-specific collagen I antibodies and in situ hybridization of collagen α1(I) gene. Pancreatic fibrosis was induced by cerulean injection and tumors by spleen injection of pancreatic tumor cells.ResultsInhibition of stroma cell infiltration and reduction of tumor ECM levels by halofuginone inhibited development of tumors derived from mouse and human pancreatic cancer cells. Halofuginone reduced the number only of stroma myofibroblasts expressing both contractile and collagen biosynthesis markers. Both stroma myofibroblasts and tumor cells generated ECM that contributes to tumor growth. Combination of treatments that inhibit stroma cell infiltration, cause apoptosis of myofibroblasts and inhibit Smad3 phosphorylation, with chemotherapy that increases tumor-cell apoptosis without affecting Smad3 phosphorylation was more efficacious than either treatment alone. More tumors developed in fibrotic than in normal pancreas, and prevention of tissue fibrosis greatly reduced tumor development.ConclusionsThe utmost importance of tissue fibrosis and of stroma cells for tumor development presents potential new therapy targets, suggesting combination therapy against stroma and neoplastic cells as a treatment of choice.

Generation of a new bioluminescent model for visualisation of mammary tumour development in transgenic mice

BackgroundNumerous transgenic models have been generated to study breast cancer. However, despite many advantages, traditional transgenic models for breast cancer are also burdened with difficulties in early detection and longitudinal observation of transgene-induced tumours, which in most cases are randomly located and occur at various time points. Methods such as palpation followed by mechanical measurement of the tumours are of limited value in transgenic models. There is a crucial need for making these previously generated models suitable for modern methods of tumour visualisation and monitoring, e.g. by bioluminescence-based techniques. This approach was successfully used in the current study.ResultsA new mouse strain (MMTV-Luc2 mice) expressing Luc2 luciferase primarily in mammary tissue in females, with low-level background expression in internal organs, was generated and bred to homozygosity. After these mice were intercrossed with MMTV-PyVT mice, all double transgenic females developed mammary tumours by the age of 10 weeks, the localisation and progression of which could be effectively monitored using the luminescence-based in vivo imaging. Luminescence-based readout allowed for early visualisation of the locally overgrown mammary tissue and for longitudinal evaluation of local progression of the tumours. When sampled ex vivo at the age of 10 weeks, all tumours derived from MMTV-Luc2PyVT females displayed robust bioluminescent signal.ConclusionsWe have created a novel transgenic strain for visualisation and longitudinal monitoring of mammary tumour development in transgenic mice as an addition and/or a new and more advanced alternative to manual methods. Generation of this mouse strain is vital for making many of the existing mammary tumour transgenic models applicable for in vivo imaging techniques.

The role of miR‐17‐92 cluster in hepatic carcinogenesis

MiR‐17‐92 cluster is a potent oncogenic miRNA which has been shown to promote cell proliferation, increase angiogenesis and sustain cell survival in several cancer types. However, the potential role of miR‐17‐92 cluster in hepatic carcinogenesis has not been investigated. This study was designed to examine the impact of miR‐17‐92 in hepatic cancer cells. qRT‐PCR analysis showed that the expression of miR‐17‐92 cluster is increased in several human hepatic cancer cell lines (Hep3B, Huh7 and HepG2) compared to the transformed, non‐cancerous hepatic cell line, THLE‐2. The miR‐17‐92 inhibitors (blocking the entire cluster or individual miRNAs within the cluster) prevented hepatic cancer cell growth. In contrast, stable transfection of pri‐miR‐17‐92 cluster increased tumor cell proliferation. Western blot analysis showed decreased PTEN protein in hepatic cancer cells transfected with the pri‐miR‐17‐92 cluster, suggesting that PTEN may represent one of the miR‐17‐92 downstream targets. Experiments are ongoing to document hepatocellular cancer development in transgenic mice with targeted expression of homo miR‐17‐92 cluster gene in the liver (driven by albumin‐cre, with or without diethylnitrosamine treatment). Results of these experiments are expected to provide important in vitro and in vivo evidences to define the role of miR‐17‐92 in hepatic carcinogenesis and tumor progression.

Live Imaging of Protein Kinase Activities in Transgenic Mice Expressing FRET Biosensors

Genetically-encoded biosensors based on the principle of Förster resonance energy transfer (FRET) have been widely used in biology to visualize the spatiotemporal dynamics of signaling molecules. Despite the increasing multitude of these biosensors, their application has been mostly limited to cultured cells with transient biosensor expression, due to particular difficulties in the development of transgenic mice that express FRET biosensors. In this study, we report the efficient generation of transgenic mouse lines expressing heritable and functional biosensors for ERK and PKA. These transgenic mice were created by the cytoplasmic co-injection of Tol2 transposase mRNA and a circular plasmid harbouring Tol2 recombination sites. High expression of the biosensors in a wide range of cell types allowed us to screen newborn mice simply by inspection. Observation of these transgenic mice by two-photon excitation microscopy yielded real-time activity maps of ERK and PKA in various tissues, with greatly improved signal-to-background ratios. Our transgenic mice may be bred into diverse genetic backgrounds; moreover, the protocol we have developed paves the way for the generation of transgenic mice that express other FRET biosensors, with important applications in the characterization of physiological and pathological signal transduction events in addition to drug development and screening.

CADASILの診断，病態，治療の進歩―本邦におけるCADASIL診断基準の作成―

During the past 10 years, our understanding of the pathomechanism and pathophysiology of cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) has improved through clinical examination, imaging studies, pathological studies, cell experiments and the development of transgenic mice. Although epidemiological studies of CADASIL in Japan have been limited, more than 100 cases of this condition have been diagnosed in Japan. In our laboratory, we diagnosed 37 CADASIL cases genetically and identified three features common to Japanese cases. One is the wide distribution of onset age for clinical symptoms other than migraine, with the onset of symptoms being later than age 60 in 22% of cases. Second, the majority (65%) of Japanese CADASIL cases have stroke risk factors, such as hypertension, hyperlipidemia, or smoking. Third, in 22% cases there was no definite family history of stroke. However, the previous diagnostic criteria proposed by Dabous excluded several definite cases in our cohort. Therefore, to avoid missing undiagnosed cases of CADASIL, we have generated new diagnostic criteria for Japanese CADASIL based on the knowledge accumulated during the past 10 years, and compared sensitivity of two criteria. In our diagnosed Japanese CADASIL cases, the sensitivity of the new criteria was 19% and 78% for probable and possible cases, respectively, and only one case was (Fig. 3) missed when using the new criteria. In comparison, the sensitivity of Dabous's was 11% and 51% for probable and possible cases, respectively, and 24% cases were excluded due to hypertension, elderly onset or no family history, although these cases showed recurrent strokes, white matter lesions and NOTCH3 mutations. Using our new criteria, diagnosis of CADASIL can be made even in cases with elderly onset, stroke risk factors, and obscure family history.

Follicular dendritic cells, conduits, lymphatic vessels, and high endothelial venules in tertiary lymphoid organs: Parallels with lymph node stroma

In this communication, the contribution of stromal, or non-hematopoietic, cells to the structure and function of lymph nodes (LNs), as canonical secondary lymphoid organs (SLOs), is compared to that of tertiary lymphoid tissue or organs (TLOs), also known as ectopic lymphoid tissues. TLOs can arise in non-lymphoid organs during chronic inflammation, as a result of autoimmune responses, graft rejection, atherosclerosis, microbial infection, and cancer. The stromal components found in SLOs including follicular dendritic cells, fibroblast reticular cells, lymphatic vessels, and high endothelial venules and possibly conduits are present in TLOs; their molecular regulation mimics that of LNs. Advances in visualization techniques and the development of transgenic mice that permit in vivo real time imaging of these structures will facilitate elucidation of their precise functions in the context of chronic inflammation. A clearer understanding of the inflammatory signals that drive non-lymphoid stromal cells to reorganize into TLO should allow the design of therapeutic interventions to impede the progression of autoimmune activity, or alternatively, to enhance anti-tumor responses.

Histological and ultrastructural comparison of cauterization and thrombosis stroke models in immune-deficient mice.

BackgroundStroke models are essential tools in experimental stroke. Although several models of stroke have been developed in a variety of animals, with the development of transgenic mice there is the need to develop a reliable and reproducible stroke model in mice, which mimics as close as possible human stroke.MethodsBALB/Ca-RAG2-/-γc-/- mice were subjected to cauterization or thrombosis stroke model and sacrificed at different time points (48hr, 1wk, 2wk and 4wk) after stroke. Mice received BrdU to estimate activation of cell proliferation in the SVZ. Brains were processed for immunohistochemical and EM.ResultsIn both stroke models, after inflammation the same glial scar formation process and damage evolution takes place. After stroke, necrotic tissue is progressively removed, and healthy tissue is preserved from injury through the glial scar formation. Cauterization stroke model produced unspecific damage, was less efficient and the infarct was less homogeneous compared to thrombosis infarct. Finally, thrombosis stroke model produces activation of SVZ proliferation.ConclusionsOur results provide an exhaustive analysis of the histopathological changes (inflammation, necrosis, tissue remodeling, scarring...) that occur after stroke in the ischemic boundary zone, which are of key importance for the final stroke outcome. This analysis would allow evaluating how different therapies would affect wound and regeneration. Moreover, this stroke model in RAG 2-/- γC -/- allows cell transplant from different species, even human, to be analyzed.

To B or not to B: Role of B cells in pathogenesis of arthritis in HLA transgenic mice

Population studies have shown that amongst all the genetic factors linked with autoimmune disease development, MHC class II genes are the most significant. Experimental autoimmune arthritis resembling human rheumatoid arthritis (RA) can be induced in susceptible strains of mice following immunization with type II collagen (CIA). We generated transgenic mice lacking endogenous class II molecules and expressing various HLA genes including RA-associated, HLA-DRB1*0401 and HLA-DQ8, and RA-resistant, DRB1*0402, genes. The HLA molecules in these mice are expressed on the cell surface and can positively select CD4+ T cells expressing various Vβ T cell receptors. Endogenous class II invariant chain is required for proper functioning of the class II transgene. Arthritis development in transgenic mice is CD4+ and B cells dependent. Studies in humanized mice showed that B cells are required as antigen presenting cells in addition to antibody producing cells for the development of CIA. The transgenic mice expressing *0401 and *0401/DQ8 genes developed sex-biased arthritis with predominantly females being affected, similar to that of human RA. Further, the transgenic mice produced autoantibodies like rheumatoid factor and anti-cyclic antibodies. Antigen presentation by B cells leads to a sex-specific immune response in DRB1*0401 mice suggesting a role of B cells and HLA-DR in rendering susceptibility to develop arthritis in females.

Developmental Stage Is a Major Determinant of Lung Injury in a Murine Model of Bronchopulmonary Dysplasia

Bronchopulmonary dysplasia (BPD) is a common inflammatory lung disease in premature infants. To study the hypothesis that the sensitivity of the lung to inflammatory injury depends on the developmental stage, we studied postnatal lung development in transgenic mice expressing human IL-1β (hIL-1β) in the lungs during the late canalicular-early saccular, saccular, or late saccular-alveolar stage. Overexpression of hIL-1β in the saccular stage caused arrest in alveolar development, airway remodeling, and goblet cell hyperplasia in the lungs as well as poor growth and survival of infant mice. Overexpression of hIL-1β during the late canalicular-early saccular stage did not adversely affect lung development, growth, or survival of the pups. Mice expressing hIL-1β from the late saccular to alveolar stage had smaller alveolar chord length, thinner septal walls, less airway remodeling and mucus metaplasia, and better survival than mice expressing hIL-1β during the saccular stage. Human IL-1β overexpression in the saccular stage was sufficient to cause a BPD-like illness in infant mice, whereas the lung was more resistant to hIL-1β-induced injury at earlier and later developmental stages.

Cooperation between Stat3 and Akt Signaling Leads to Prostate Tumor Development in Transgenic Mice

In this report, we describe the development of a transgenic mouse in which a rat probasin promoter (ARR(2)Pb) was used to direct prostate specific expression of a constitutively active form of signal transducer and activator of transcription 3 (i.e., Stat3C). ARR(2)Pb.Stat3C mice exhibited hyperplasia and prostate intraepithelial neoplasia (PIN) lesions in both ventral and dorsolateral prostate lobes at 6 and 12 months; however, no adenocarcinomas were detected. The effect of combined loss of PTEN was examined by crossing ARR(2)Pb.Stat3C mice with PTEN(+/-) null mice. PTEN(+/-) null mice on an ICR genetic background developed only hyperplasia and PIN at 6 and 12 months, respectively. ARR(2)Pb.Stat3C x PTEN(+/-) mice exhibited a more severe prostate phenotype compared with ARR(2)Pb.Stat3C and PTEN(+/-) mice. ARR(2)Pb.Stat3C x PTEN(+/-) mice developed adenocarcinomas in the ventral prostate as early as 6 months (22% incidence) that reached an incidence of 61% by 12 months. Further evaluations indicated that phospho-Stat3, phospho-Akt, phospho-nuclear factor κB, cyclin D1, and Ki67 were upregulated in adenocarcinomas from ARR(2)Pb.Stat3C x PTEN(+/-) mice. In addition, membrane staining for β-catenin and E-cadherin was reduced. The changes in Stat3 and nuclear factor κB phosphorylation correlated most closely with tumor progression. Collectively, these data provide evidence that Stat3 and Akt signaling cooperate in prostate cancer development and progression and that ARR(2)Pb.Stat3C x PTEN(+/-) mice represent a novel mouse model of prostate cancer to study these interactions.

Abstract 2476: Tumor development in transgenic mice constitutively expressing hypoxia-inducible factor-1α

Abstract Hypoxia-inducible factor-1α (HIF-1α) has an essential transcription factor for regulation of various genes associated with hypoxic condition in tissues. Elevated expression of HIF-1α has been reported in many cancers associated with tumor progression, invasion and metastasis. To investigate the role of HIF-1α in tumor development and metastasis, we established transgenic mice constitutively expressing HIF-1α gene under regulation of CMV promoter. The transgenic mice showed no gross morphological abnormality until 3 months after birth, but the mice developed tumors in lymphoid, lung and breast tissues. The incidence of tumor bearing in transgenic mice was up to 90% until 18 months after birth. Expression of HIF-1α mRNA and protein was varied among tissues, but the amount of HIF-1α gene was gradually increased in an age-dependent manner. To clarify the molecular mechanism of lymphomagenesis in the transgenic mice, subpopulation of the lymphocytes, mitogenic responsiveness and cytokine production on lymphocyte from transgenic mice were analyzed. We next analyzed the expression of HIF-1α in human lymphoma tissues and found apparent expression of the gene in angioimmunoblastic T cell lymphoma (AILT). Since VEGF expression and marked increase of small vessels in tumor tissues are common features in AILT, HIF-1α may play an important role in pathogenesis of the lymphoma. We present the results concerning the role of HIF-1α in lymphoma occurrence, and will discuss the contribution of the transcription factor in human cancer development. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2476.

The role of GABA-A receptor subtypes as analgesic targets

Recent GABA(A) receptor drug discovery efforts have culminated in the development of transgenic mice and subtype-selective pharmacological tools, enhancing our understanding of the major inhibitory neural system in the central nervous system. Notably, subtype-selective tools have demonstrated in both preclinical studies and, to some extent, in man that it is possible to develop drugs that share the clinical benefits of benzodiazepines (e.g., anxiolysis) while obviating some adverse effects of these clinically important drugs. Here, we highlight chronic pain as another therapeutic area in which subtype-selective GABA(A) receptor drugs might have clinical utility. Specifically, based on research in animal models of inflammatory/neuropathic pain, we suggest that subtype-selective positive modulators of GABA(A) alpha(2/3) receptors might reverse a loss of postsynaptic GABA(A) receptor-mediated inhibitory function in spinal cord, leading to analgesia. However, alteration of presynaptic inhibitory neural transmission in chronic pain suggests that drugs that negatively modulate GABA(A) receptors might also be effective analgesics. For example, the non-selective negative allosteric modulator FG-7142 reverses allodynia in an animal model of neuropathic pain. Importantly, these two mechanisms are not mutually exclusive. Further clinical exploration in pain of available positive and negative subtype-selective modulators that have been administered to humans would considerably aid back translation, allowing for improved therapeutic development.

Matrix Metalloproteinase-9 Deficiency Worsens Lung Injury in a Model of Bronchopulmonary Dysplasia

Increased activity of matrix metalloproteinase (MMP)-9 is associated with the development of bronchopulmonary dysplasia (BPD) in newborn infants, but the role of MMP-9 in the pathophysiology of BPD is unclear. We have shown that perinatal expression of interleukin-1 beta (IL-1 beta) in the lung is sufficient to cause a BPD-like illness in infant mice. To study the hypothesis that MMP-9 is an important downstream mediator in IL-1 beta-induced lung injury in the newborn, we compared the effects of IL-1 beta on fetal and postnatal lung inflammation and development in transgenic mice with regulatable pulmonary overexpression of human mature IL-1 beta with wild-type (IL-1 beta/MMP-9(+/+)) or null (IL-1 beta/MMP-9(-/-)) MMP-9 loci. IL-1 beta increased the expression of MMP-9 mRNA and amount of MMP-9 protein in the lungs of MMP-9(+/+) mice. IL-1 beta/MMP-9(-/-) mice had fewer neutrophils but more macrophages in the lungs than did IL-1 beta/MMP-9(+/+) mice. MMP-9 deficiency increased pulmonary cell death and macrophage clearance of dying cells in IL-1 beta-expressing mice. IL-1 beta/MMP-9(-/-) mice had more severe alveolar hypoplasia than IL-1 beta/MMP-9(+/+) mice, implying that IL-1 beta-induced lung disease was worsened in the absence of MMP-9. These results suggest that MMP-9 activity in the inflamed neonatal lung protects the lung against injury.

Genetically modified mouse models in cancer studies

Genetically modified animals represent a resource of immense potential for cancer research. Classically, genetic modifications in mice were obtained through selected breeding experiments or treatments with powerful carcinogens capable of inducing random mutagenesis. A new era began in the early 1980s when genetic modifications by inserting foreign DNA genes into the cells of an animal allowed for the development of transgenic mice. Since that moment, genetic modifications have been able to be made in a predetermined way. Gene targeting emerged later as a method of in vivo mutagenesis whereby the sequence of a predetermined gene is selectively modified within an intact cell. In this review we focus on how genetically modified mice can be created to study tumour development, and how these models have contributed to an understanding of the genetic alterations involved in human cancer. We also discuss the strengths and weaknesses of the different mouse models for identifying cancer genes, and understanding the consequences of their alterations in order to obtain the maximum benefit for cancer patients.

E2F1 Inhibits c-Myc-Driven Apoptosis via PIK3CA/Akt/mTOR and COX-2 in a Mouse Model of Human Liver Cancer

Resistance to apoptosis is essential for cancer growth. We previously reported that hepatic coexpression of c-Myc and E2F1, 2 key regulators of proliferation and apoptosis, enhanced hepatocellular carcinoma (HCC) development in transgenic mice. Here, we investigated the molecular mechanisms underlying oncogenic cooperation between c-Myc and E2F1 in relationship to human liver cancer. Activation of pro- and antiapoptotic cascades was assessed by immunoblotting in experimental HCC models and in human HCC. Effect of antisense oligodeoxy nucleotides against c-Myc and E2F1 was studied in human HCC cell lines. Suppression of catalytic subunit p110alpha of phosphatidylinositol 3-kinase (PIK3CA)/Akt, mammalian target of rapamycin (mTOR), and cyclooxygenase (COX)-2 pathways was achieved by pharmacologic inhibitors and small interfering RNA in human and mouse HCC cell lines. Coexpression with E2F1 did not increase proliferation triggered by c-Myc overexpression but conferred a strong resistance to c-Myc-initiated apoptosis via concomitant induction of PIK3CA/Akt/mTOR and c-Myb/COX-2 survival pathways. COX-2 was not induced in c-Myc and rarely in E2F1 tumors. In human HCC, PIK3CA/Akt/mTOR and c-Myb/COX-2 pathways were similarly activated, with levels of PIK3CA/Akt, mTOR, and c-Myb being inversely associated with patients' survival length. Silencing c-Myc and E2F1 reduced PIK3CA/Akt and mTOR and completely abolished c-Myb and COX-2 expression in human HCC cell lines. Finally, simultaneous inhibition of PIK3CA/Akt/mTOR and COX-2 activity in in vitro models caused massive apoptosis of neoplastic hepatocytes. E2F1 may function as a critical antiapoptotic factor both in human and in rodent liver cancer through its ability to counteract c-Myc-driven apoptosis via activation of PIK3CA/Akt/mTOR and c-Myb/COX-2 pathways.

Roles of induced expression of MAPK phosphatase-2 in tumor development in RET-MEN2A transgenic mice

Germline mutations in the RET tyrosine kinase gene are responsible for the development of multiple endocrine neoplasia 2A and 2B (MEN2A and MEN2B). However, knowledge of the fundamental principles that determine the mutant RET-mediated signaling remains elusive. Here, we report increased expression of mitogen-activated protein kinase phosphatase-2 (MKP-2) in carcinomas developed in transgenic mice carrying RET with the MEN2A mutation (RET-MEN2A). The expression of MKP-2 was not only induced by RET-MEN2A or RET-MEN2B mutant proteins but also by the activation of endogenous RET by its ligand, glial cell line-derived neurotrophic factor (GDNF). MKP-2 expression was also evident in the MKK-f cell line, which was established from a mammary tumor developed in a RET-MEN2A transgenic mouse. Inhibition of MKP-2 attenuated the in vitro and in vivo proliferation of MKK-f cells, which was mediated by the suppression of cyclin B1 expression. Furthermore, we found that MKP-2 is highly expressed in medullary thyroid carcinomas derived from MEN2A patients. These findings suggest that the increased expression of MKP-2 may play a crucial role in oncogenic signaling downstream of mutant RET, leading to deregulation of cell cycle.