Characterization of adrenocortical cell lines produced by genetically targeted tumorigenesis in transgenic mice

Microvascular hyperaemia is decreased in subjects at risk of developing non-insulin-dependent diabetes mellitus (NIDDM) who have fasting hyperglycaemia. Such microvascular abnormalities may be involved in the pathogenesis of diabetic microangiopathy. To investigate the relationship of reduced microvascular hyperaemia to metabolic and blood pressure abnormalities associated with the prediabetic state, we studied 24 subjects with fasting hyperglycaemia and 24 age- and sex-matched control subjects. The microvascular hyperaemic response to local heating of the skin on the dorsum of the foot measured by laser Doppler fluximetry was reduced in the subjects with fasting hyperglycaemia (1.18 [0.87-1.83] volts vs 1.51 [1.30-2.14] volts normal subjects; p = 0.0002) and was negatively correlated with fasting plasma insulin concentration (Rs = 0.70; p = 0.001) and positively related to insulin sensitivity determined by continuous infusion of glucose with model assessment (CIGMA) (Rs = 0.52; p = 0.01), but showed no association with fasting plasma glucose, beta-cell function 24 h ambulatory blood pressure profiles or serum lipid concentrations. These results suggests that hyperinsulinaemia, as a result of insulin resistance, may have a detrimental effect on microvascular function in the prediabetic state.     

Immortalization of neuroendocrine pinealocytes from transgenic mice by targeted tumorigenesis using the tryptophan hydroxylase promoter

Tryptophan hydroxylase (TPH) is the first enzyme in both serotonin and melatonin biosynthesis in neuroendocrine cells of the pineal gland. The lack of immortalized neuroendocrine pineal cell lines has been a major obstacle to the study of the tissue-specific and circadian regulation of TPH gene expression in the pineal gland. Previously, we demonstrated that a 6.1 kb 5' upstream region of the mouse TPH gene directs the restricted expression of a lacZ reporter gene to the pineal gland and the raphe nuclei of transgenic mice. Therefore, to develop TPH-expressing pineal cell lines we first established transgenic mice carrying a construct consisting of 6.1 kb of 5' flanking region fused to the SV40 T-antigen. These animals developed highly invasive pineal tumors and died at 12-15 weeks of age. The pineal tumors obtained from the transgenic mice were utilized to establish the immortalized pinealocyte-derived cell lines. These cells express two marker enzymes, TPH and serotonin N-acetyltransferase (NAT). In pineal gland TPH and NAT expressions have been known to be regulated during circadian cycle. The two established cell lines therefore promise to be a valuable in vitro model system for the study of the rhythmic nature of the pineal function at molecular level in mammal.     

Genomic imprinting and Igf2 influence liver tumorigenesis and loss of heterozygosity in SV40 T antigen transgenic mice

Maternal-specific loss of heterozygosity (LOH) and allelic imbalances [i.e., partial LOH (pLOH)] observed in SV40 T/t antigen-induced liver tumors suggests that an imprinted gene on chromosome 7 is involved in liver tumorigenesis. Maternal-specific LOH/pLOH may reflect the loss of a maternally expressed tumor suppressor gene or the acquisition of paternally active alleles of a growth promoter. In addition, two oppositely imprinted genes on distal chromosome 7, Igf2 and H19, are re-expressed in most liver tumors from an SV40 T/t antigen transgenic line (M11T-G). Igf2 is a paternally expressed growth promoter, and H19 is a maternally expressed gene that can suppress growth in some tumor cell lines. We studied the role of Igf2 during liver tumorigenesis by creating Igf2 (+/-) M11T-G mice. These mice are essentially null for Igf2 expression because imprinting normally precludes maternal Igf2 expression. M11T-G, Igf2 (+/-) males exhibit a 15-fold reduction in the frequency of large tumors. Igf2 (+/-) tumors do not express maternal Igf2, indicating rigid imprinting control in the liver. LOH/pLOH analysis was performed on the tumors and indicates that acquisition of paternally active Igf2 alleles is a major selective event for M11T-G liver tumorigenesis. This also implies the existence of an imprinted, maternally expressed tumor suppressor gene on chromosome 7 that is unlikely to be H19.     

Islet regeneration in IFNgamma transgenic mice

The adult pancreas is a developmentally stable organ with limited mitotic activity. This minimal mitotic activity proves critical in insulin-dependent diabetes mellitus (IDDM) since the pathogenesis is characterized by a selective and permanent destruction of islet beta cells. The IFNgamma transgenic mouse model of islet regeneration elucidates the differentiation pathway involved in the regeneration of functional beta cells from ductal precursors and reveals the functional plasticity of the adult pancreas.     

Effect of the viable-yellow (A(vy)) agouti allele on skin tumorigenesis and humoral hypercalcemia in v-Ha-ras transgenic TGxAC mice

We previously reported that papillomas can arise from the follicular epithelium of v-Ha-ras transgenic TGxAC mice. Since the viable-yellow mutation (A(vy)) of the mouse agouti gene which regulates coat color pigmentation by acting within the micro-environment of the hair follicle has been shown to function as a tumor promoter in the liver, we hypothesized that it may also play a role in TGxAC skin tumorigenesis. Endogenous agouti protein product was detected in the outer root sheath of anagen hair follicles following plucking of the hair shaft, but not in the interfollicular epithelium, in TGxAC mice on an FVB/N genetic background. It was also detected in papillomas from these mice produced by 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment or plucking. Expression of the A(vy) allele in the v-Ha-ras transgenic TGxAC mouse line results in an approximately 2-fold increase in papilloma development compared with controls which did not carry the A(vy) allele following twice-weekly treatment with 1.25, 2.5 or 5.0 microg TPA. In addition, TPA-treated, papilloma-bearing F1 mice which carried the A(vy) allele, but not F1 mice which did not carry the A(vy) allele, exhibited a syndrome of humoral hypercalcemia mediated by parathyroid hormone-related protein (PTHrP) that led to weight loss, hypercalcemia and hypophosphatemia. Thus, we conclude that the A(vy) allele can influence the development of skin tumors and PTHrP-mediated humoral hypercalcemia in v-Ha-ras transgenic TGxAC mice.     

Inhibition of hepatitis B virus replication during adenovirus and cytomegalovirus infections in transgenic mice

We have previously demonstrated that hepatitis B virus (HBV) replication and gene expression are abolished in the livers of HBV transgenic mice by cytotoxic T lymphocytes (CTLs) and during lymphocytic choriomeningitis virus (LCMV) infection, stimuli that trigger the production of alpha/beta interferon, gamma interferon, and tumor necrosis factor alpha in the liver. We now report that hepatic HBV replication and gene expression are inhibited by the local induction of these cytokines during adenovirus- and murine cytomegalovirus (MCMV)-induced hepatitis. Further, we show that MCMV also blocks HBV replication and gene expression in the proximal convoluted tubules of the kidney by causing interstitial nephritis and inducing the same cytokines in the renal parenchyma. These results suggest that inflammatory cytokines probably contribute to viral clearance during acute viral hepatitis in humans, and they imply that induction of these cytokines in the liver and other infected tissues of chronically infected patients might have therapeutic value.     

Morphological study of pituitary tumorigenesis in transgenic mice induced by hybrid oncogene of the thyrotropin beta-subunit and the simian virus 40 large T-antigen

We have created a transgenic mouse, TTP-1, generating anterior pituitary tumors by using the simian virus 40 (SV40) large T antigen gene and human thyrotropin beta-subunit gene. To examine characteristics of tumors, histological details were investigated using light and electron microscopies. The main tumor tissues, composed of small chromophobe cells, were located inferior to but clearly separated from the hypothalamus; however, neuron fibers probably derived from the hypothalamus were observed to invade some tumor tissues. Some differentiated endocrine cells occupied the caudal region of the tumor. Immunohistochemically, SV40 large T antigen was expressed in the cell nucleus of the undifferentiated cell area, whereas cells expressing several hormones were mainly distributed in the differentiated cell area. Electron microscopically, the undifferentiated cells were divided into 2 types; electron-dense and -lucent cells, the nuclei of which were composed of obscured nucleoli and many notable invaginations of the nuclear membrane. No intracellular microfilamentous structures were observed. Sometimes it was noted that cytoplasmic processes were connected with gap junctions. In the intercellular spaces, there were neuron fibrous and synapse-like structures. In the differentiated cell area, the cell membranes directly contacting other cells were relatively smooth, and many gap junctions were demonstrated. Secretory granules, which were round and less than 100 nm in diameter, were more electron dense in smaller cells than in larger cells. They were aligned just below the cell membrane. Immuno-electron microscopically, positive reactions for SV40 were observed in the nuclei of the undifferentiated cell area. In the differentiated cell area, most of the secretory granules were labeled by GH. TTP-1 transgenic mice should provide a valuable animal model for studying the pathogenesis of anterior pituitary tumors.     

Lessons from TGF-beta transgenic mice

Aberrant expression of TGF-beta and/or receptor/signaling function is present in a wide variety of disease processes. Overexpression of TGF-beta isoforms in transgenic mice using tissue-specific promoters has provided model systems to study the effects of increased activity of TGF-beta in the intact organism. We will review the pertinent features of some of these models, and discuss new insights provided by these studies into regulation and role of TGF-beta in health and disease.     

Active signaling by Neu in transgenic mice

Transgenic mice engineered to overexpress the HER-2/neu/erbB-2 protooncogene under the control of a mammary-specific promoter develop mammary tumors and are a model for human breast cancer. Signal transduction by Neu was examined in situ in the tumors of these transgenic mice. This was accomplished using the PN2A monoclonal antibody, which recognizes Neu only in the phosphorylated, and therefore actively signaling, state. Immunohistochemistry using PN2A demonstrated that Neu actively signals in the tumors of Neu transgenic mice. Expression of Neu was always accompanied by co-overexpression of the endogenous epidermal growth factor receptor. Qualitatively similar results were found in mammary tumors from mice bitransgenic for the neu and transforming growth factor-alpha genes (both driven by the mouse mammary tumor virus promoter). Early mammary lesions demonstrated distinctive patterns of Neu activation relative to expression levels. Overexpression and activation were separable both temporally and spatially. These results refine the multi-step model for the role of Neu in mammary neoplasia and establish phosphorylation-state specific antibodies as a powerful tool for investigating tumor progression.     

Osteochondrodysplasia occurring in transgenic mice expressing interferon-gamma

Arterial thrombi are primarily composed of platelets. Platelets are bound to injured endothelial cells, sub-endothelial matrices, and other platelets by a range of adhesive proteins. Some of these reactions are governed by shear forces. The role of adhesive proteins in the pathogenesis of arterial thrombosis is not fully understood. The aim of this study was to examine the involvement of von Willebrand factor (vWF), fibrinogen (Fg), and fibronectin (FN) in the formation of microvascular thrombi in vivo using a helium-neon laser-induced thrombosis method. Transmission electron microscopy demonstrated that laser irradiation resulted in platelet-rich thrombosis in arterioles and venules, and revealed that this occurred in the absence of endothelial denudation. The mean wall shear rates in mesenteric arterioles and venules were 641 +/- 40 and 280 +/- 20 s-1, respectively. Shear rates increased approximately fivefold in arterioles and tenfold in venules during the formation of occlusive thrombi. Antibody to vWF inhibited thrombosis in arterioles and venules. Antibodies to Fg and FN inhibited thrombosis in venules but not in arterioles. These results confirm that vWF, Fg and FN were involved in thrombogenesis in vivo and demonstrated that significantly higher shear rates were required for the reactions involving vWF than those involving either Fg or FN.     

Primary demyelination in transgenic mice expressing interferon-gamma

Ever since the use of interferon-gamma to treat patients with multiple sclerosis resulted in enhanced disease, the role of IFN-gamma in demyelination has been under question. To address this issue directly, transgenic mice were generated that expressed the cDNA of murine IFN-gamma in the central nervous system by using an oligodendrocyte-specific promoter. Expression of the transgene occurred after 8 weeks of age, at which time the murine immune and central nervous systems are both fully developed. Directly associated with transgene expression, primary demyelination occurred and was accompanied by clinical abnormalities consistent with CNS disorders. Additionally, multiple hallmarks of immune-mediated CNS disease were observed including upregulation of MHC molecules, gliosis and lymphocytic infiltration. These results demonstrate a direct role for IFN-gamma as an inducer of CNS demyelination leading to disease and provide new opportunities for dissecting the mechanism of demyelination.     

Targeted overexpression of Bcl-2 in ovaries of transgenic mice leads to decreased follicle apoptosis, enhanced folliculogenesis, and increased germ cell tumorigenesis

We have generated transgenic mice overexpressing Bcl-2, an apoptosis suppression protein, in ovarian cells using the inhibit-alpha gene promoter/enhancer. Ovarian apoptotic DNA fragmentation induced in immature animals by a low dose (2 IU) of PMSG was suppressed by greater than 55% in transgenic mice compared to their wild-type littermates. Morphological and in situ DNA end-labeling analyses showed that granulosa cells in large antral follicles of wild-type animals undergo apoptosis, but most follicles in transgenic animals are healthy. When the animals were treated with a high dose (4 IU) of PMSG to stimulate follicular growth, spontaneous ovulation was observed in 14 of 23 (61%) of the transgenic animals, but in only 3 of 18 (17%) of wild-type siblings. Furthermore, transgenic females had a larger litter size (9.07 +/- 0.25 pups/litter; n = 29) than wild-type controls (7.54 +/- 0.26 pups/litter; n = 28; P < 0.01). These data suggested that decreased ovarian apoptosis in transgenic animals could lead to enhanced folliculogenesis and ovulatory potential. Moreover, aging transgenic mice are susceptible to the development of benign cystic ovarian teratoma (4 in 20 transgenic animals and 0 in 26 wild-type controls). Some tumor tissues showed respiratory and intestinal cell types, whereas others showed the development of central nervous system-like structures. Because the bcl-2 transgene in these animals is overexpressed in somatic cells, but not oocytes, these findings suggest that enhanced survival of selected somatic cells in transgenic mice could lead to germ cell tumorigenesis. Thus, overexpression of Bcl-2 protein in the ovary leads to decreased ovarian somatic cell apoptosis, enhanced folliculogenesis, and increased susceptibility to ovarian germ cell tumorigenesis in transgenic animals. The present mouse model allows future studies on intracellular signal pathways regulating follicular atresia and on the potential role of ovarian somatic cell factors in germ cell tumorigenesis.     

Behavioural phenotyping of transgenic mice.

This paper reviews the current work on mouse genetics, brain, and behaviour in my laboratory. It starts with an historical account of our research and shows how certain research themes, such as olfaction, learning, social behaviour, and environmental effects in rodents have led to our current research on behavioural phenotyping of inbred, mutant, knockout, and transgenic mice. We are concerned with finding neural and behavioural sequelae to genetic manipulations in mice and use a battery of tests to detect behaviours that are altered in genetically modified mice. In this way we are working to dissociate neural and behavioural effects of different gene manipulations in mouse models of neurodegenerative diseases. Sensory, motor, cognitive, affective, and social behaviours may all be affected by gene manipulation, thus careful behavioural techniques, with attention to the mice themselves, the apparatus, and procedure, experimenter variables, and environmental effects are necessary in order to determine a reliable and valid mouse behavioural phenotype. As both the genome and the environment have significant effects on the behavioural phenotype, our future research will utilize an epigenetic approach to examine how environmental cues modulate gene expression in the behavioural phenotyping of transgenic mice. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Apoptosis of cardiac myocytes in Gsalpha transgenic mice

We examined the potential involvement of two CC chemokine receptors (CCRs), CCR-1 and CCR-3, in the functional activation of granulocyte-macrophage colony-stimulating factor (GM-CSF) plus interleukin-4 (IL-4)-generated human peripheral blood monocyte-derived immature dendritic cells (DCs). Flow cytometric analysis showed that CCR-1, CCR-3, CCR-5, and CXC chemokine receptor (CXCR)-4 were expressed on the cell surface of monocyte-derived DCs. Treatment with a monoclonal antibody (MoAb) to either CCR-1 or CCR-3 but not MoAbs to CCR-5 and CXCR-4 abolished chemotactic migration of monocyte-derived DCs. The DCs treated with either the anti-CCR-1 MoAb or anti-CCR-3 MoAb were less efficient than untreated DCs in proliferation of allogeneic T cells (TCs) and TC-derived secretion of interferon-gamma (IFN-gamma). The homotypic aggregation of DCs and heterotypic aggregation of DCs with TCs were suppressed by the anti-CCR-1 MoAb or anti-CCR-3 MoAb. These results indicate that CCR-1 and CCR-3 specifically regulate interaction of TCs and DCs in the process of antigen presentation.     

Angiogenesis and apoptosis are cellular parameters of neoplastic progression in transgenic mouse models of tumorigenesis

The epidemiology and histopathology of human cancers and studies of animal models of tumorigenesis have led to a widely-accepted notion that multiple genetic and epigenetic changes have to accrue for the successful development of a malignant phenotype. Tumor growth and expansion requires an ability not only to proliferate, but also to down-modulate cell death (apoptosis) and activate angiogenesis to produce a tumor neovasculature. This review will describe the interplay between apoptosis and proliferation, as well as the characteristics of the angiogenic phenotype in two transgenic mouse models of multi-step tumorigenesis, namely, pancreatic islet cell carcinomas and squamous cell carcinomas of the skin.     

Hyperplasia of lymphatic vessels in VEGF-C transgenic mice

No growth factors specific for the lymphatic vascular system have yet been described. Vascular endothelial growth factor (VEGF) regulates vascular permeability and angiogenesis, but does not promote lymphangiogenesis. Overexpression of VEGF-C, a ligand of the VEGF receptors VEGFR-3 and VEGFR-2, in the skin of transgenic mice resulted in lymphatic, but not vascular, endothelial proliferation and vessel enlargement. Thus, VEGF-C induces selective hyperplasia of the lymphatic vasculature, which is involved in the draining of interstitial fluid and in immune function, inflammation, and tumor metastasis. VEGF-C may play a role in disorders involving the lymphatic system and may be of potential use in therapeutic lymphangiogenesis.     

Multigenic and imprinting control of ovarian granulosa cell tumorigenesis in mice

Spontaneous juvenile ovarian granulosa cell (GC) tumors that occur in young girls are similar to GC carcinomas that develop in SWR-derived inbred mice. We analyzed female offspring from a series of matings among SWR and SJL inbred mice for chromosomal loci underlying tumor susceptibility. Intercross F2 female mice were produced by reciprocal matings of (SWR x SJL)F1 and (SJL x SWR)F1 parents. Tumorigenesis in these F2 mice as well as in SWXJ recombinant inbred and congenic strains of mice derived from SWR and SJL showed significant (P < 0.001) association with Gct1, a dominant susceptibility locus on chromosome (CHR) 4 and with Gct2 on CHR 12. Suggestive (P < 0.01) association was found with Gct3 on CHR 15. A fourth susceptibility locus, Gct4 on CHR X, was demonstrated with a strong parent-of-origin effect associated with the paternal genotype. Imprinting and complex interactions among these four loci combine to establish the probability for GC tumorigenesis in this mouse model.