Relationship between expression of epidermal growth factor and simian virus 40 T antigen in a line of transgenic mice

The pattern of expression of the simian virus 40 (SV40) T antigen gene and resultant dysplasia were re-examined in a line of transgenic mice in which the T antigen gene was under the control of the SV40 early promoter. We found that T antigen expression in the kidney, and resulting dysplastic lesions, occurred exclusively in the distal convoluted tubules and the ascending limbs of Henle. Epidermal growth factor (EGF) expression in the kidney of normal mice was similarly immunolocalized. The correlation between high EGF immunoreactivity in normal mouse tissues and T antigen expression in the transgenic counterpart was also seen in the choroid plexus epithelium and in the submandibular glands of male mice. T antigen was not found in the submandibular gland of transgenic females. Similarly, EGF was only rarely detected in the normal female submandibular gland. In contrast to the correlation between T antigen expression in the transgenic mice and EGF expression in the corresponding tissues of the normal mice, within the dysplastic lesions of the transgenic mice EGF expression was severely diminished. Adenocarcinomas of the male submandibular gland from another line of transgenic mice that expresses theInt-1 transgene, showed similarly reduced levels of immunostaining for EGF. Thus, reduced expression of EGF might be a general feature of dysplasia and tumorigenesis in those tissues that normally express EGF.     

An Immortal Cell Culture Model of Hypothalamic Gonadotropin-Releasing Hormone Neurons

The neuroendocrine hypothalamus has been the object of intensive study in vivo and in tissue slices. However, using these models it is difficult to approach questions at the molecular and cellular level and to differentiate between direct effects and those mediated by other neurons. By using the regulatory domain of the rat gonadotropin-releasing hormone (GnRH) gene to target expression of the oncogene SV40 T antigen in transgenic mice, we have produced hypothalamic tumors which were cultured to produce clonal cell lines (GT1 cells). These cells express GnRH and many other neuronal markers, but do not express glial cell markers or other hypothalamic hormones. They have a distinctive neuronal phenotype, process the GnRH peptide accurately, and secrete GnRH in a pulsatile pattern. They respond to many neurotransmitters and neuromodulators including activin, norepinephrine, dopamine, nitric oxide, NMDA, and GABA, as well as GnRH itself. Thus, we have immortalized GnRH neurons by targeting oncogenesis to a defined population of neurons using the regulatory region of a gene which is expressed late in the differentiation of that cell lineage. The GT1 cell lines serve as an excellent model for molecular, pharmacological, electrophysiological, and biochemical investigations into the regulation of GnRH and the characteristics of a pure CNS neuronal population. Moreover, their derivation demonstrates the success of targeting tumorigenesis to specific differentiated neurons of the central nervous system in transgenic mice.     

Tet-on诱导表达c-myc和SV40Tag的转基因小鼠肿瘤模型

目的研究Tet-on诱导表达c-myc和SV40Tag小鼠肿瘤模型的肿瘤发生和基因表达情况,探讨c-myc基因的作用。方法用pTRE2-c-myc单阳性转基因小鼠和Tet-on、pTRE2-SV40Tag双阳性转基因小鼠交配,后代检测得到Tet-onp、TRE2-SV40Tag、pTRE2-c-myc三阳性转基因小鼠,经强力霉素诱导一段时间以后,观察肿瘤的发生;通过RT-PCR、病理组织切片和磁共振等方法对肿瘤的发生部位和时相进行研究。结果Tet-on、pTRE2-SV40Tag、pTRE2-c-myc三阳性转基因小鼠①经诱导后发生肿瘤,且发瘤率和发瘤时间高于和短于Tet-on、pTRE2-SV40Tag双阳性转基因小鼠;②c-myc和SV40Tag基因在表达部位上有所不同。结论c-myc和SV40Tag基因同时表达与SV40Tag基因单独表达时相比,肿瘤发生明显增强,提示c-myc基因与肿瘤的发生有着密切关系。     

Ets2 in Tumor Fibroblasts Promotes Angiogenesis in Breast Cancer

Tumor fibroblasts are active partners in tumor progression, but the genes and pathways that mediate this collaboration are ill-defined. Previous work demonstrates that Ets2 function in stromal cells significantly contributes to breast tumor progression. Conditional mouse models were used to study the function of Ets2 in both mammary stromal fibroblasts and epithelial cells. Conditional inactivation of Ets2 in stromal fibroblasts in PyMT and ErbB2 driven tumors significantly reduced tumor growth, however deletion of Ets2 in epithelial cells in the PyMT model had no significant effect. Analysis of gene expression in fibroblasts revealed a tumor- and Ets2-dependent gene signature that was enriched in genes important for ECM remodeling, cell migration, and angiogenesis in both PyMT and ErbB2 driven-tumors. Consistent with these results, PyMT and ErbB2 tumors lacking Ets2 in fibroblasts had fewer functional blood vessels, and Ets2 in fibroblasts elicited changes in gene expression in tumor endothelial cells consistent with this phenotype. An in vivo angiogenesis assay revealed the ability of Ets2 in fibroblasts to promote blood vessel formation in the absence of tumor cells. Importantly, the Ets2-dependent gene expression signatures from both mouse models were able to distinguish human breast tumor stroma from normal stroma, and correlated with patient outcomes in two whole tumor breast cancer data sets. The data reveals a key function for Ets2 in tumor fibroblasts in signaling to endothelial cells to promote tumor angiogenesis. The results highlight the collaborative networks that orchestrate communication between stromal cells and tumor cells, and suggest that targeting tumor fibroblasts may be an effective strategy for developing novel anti-angiogenic therapies.     

Vasopressin-SV40 T antigen expression in transgenic mice induces brain tumor and lymphoma

In order to understand the importance of various cis-acting elements in regulating VP gene expression, transgenic mice regulated by VP constructs were produced containing 3.8 kb of the 5' flanking region and all the exons and introns in the mouse VP gene, which was fused at the end of exon 3 to an SV40 T antigen (Tag). In the transgenic mice by the pVPSV.IGR3.6 construct, all the six transgenic mice died at the age of 2-6 weeks. In the transgenic mice by pVPSV.IGR2.1, 21% of them had brain tumors at 5 weeks and 100% of the mice had brain tumors after 24 weeks. Histological analysis of the transgenic mice revealed primitive neuroectodermal tumors (PNET) in the brain and lymphoma in the spleen and lymph nodes. The phenotype differences between the two transgenic mice suggest that tissue-specific expression might be regulated by cis-acting elements in the 1.5-kb of the 3(') flanking region, which are not contained in pVPSV.IGR2.1. In conclusion, pVPSV.IGR2.1 mice will be a valuable mouse model system for investigating PNET tumorigenesis in the brain and lymphoma in the lymph nodes and spleen.     

Effect of an enhanced CaMV 35S promoter and a fruit-specific promoter on uida gene expression in transgenic tomato plants

Summary Two different promoters, a cauliflower mosaic virus (CaMV) 35S promoter with a 5′-untranslated leader sequence from alfalfa mosaic virus RNA4 (designated as CaMV 35S/AMV) and an E-8 fruit-ripening-specific promoter, were compared to evaluate their effects on expression of the uidA reporter gene in transgenic tomato plants. In order to generate sufficient numbers of transgenic tomato plants, both a reliable regeneration system and an efficient Agrobacterium transformation protocol were developed using 8-d-old cotyledons of tomato (Lycopersicon ecsulentum Mill. cv. Swifty Belle). Two sets of constructs, both derivatives of the binary vector pBI121, were used in transformation of tomato whereby the uidA gene was driven either by the CaMV 35S/AMV or the E-8 fruit-ripening-specific promoter. Southern blot hybridization confirmed the stable integration of the chimeric uidA gene into the tomato genome. Fruit and leaf tissues were collected from T₀ and T₁ plants, and assayed for β-glucuronidase (GUS) enzyme activity. As expected, both vegetative and fruit tissues of transgenic plants carrying the uidA gene under the control of CaMV 35S/AMV showed varying levels of GUS activity, while no expression was observed in vegetative tissues of transgenic plants carrying the uidA gene driven by the E-8 promoter. All fruits from transgenic plants produced with both sets of constructs displayed expression of the uidA gene. However, when this reporter gene was driven by the CaMV 35S/AMV, GUS activity levels were significantly higher than when it was driven by the E-8 fruit-specific promoter. The presence/absence of the uidA gene in T₁ plants segregated in a 3∶1 Mendelian ratio.     

The Influence of SV40 polyA on Gene Expression of Baculovirus Expression Vector Systems

The simian virus 40 polyadenylation signal (SV40 polyA) has been routinely inserted downstream of the polyhedrin promoter in many baculovirus expression vector systems (BEVS). In the baculovirus prototype Autographa californica multiple nucleopolyhedrovirus (AcMNPV), the polyhedrin promoter (very late promoter) transcribes its gene by a viral RNA polymerase therefore there is no supporting evidence that SV40 polyA is required for the proper gene expression under the polyhedrin promoter. Moreover, the effect of the SV40 polyA sequence on the polyhedrin promoter activity has not been tested either at its natural polyhedrin locus or in other loci in the viral genome. In order to test the significance of adding the SV40 polyA sequence on gene expression, the expression of the enhanced green fluorescent protein (egfp) was evaluated with and without the presence of SV40 polyA under the control of the polyhedrin promoter at different genomic loci (polyherin, ecdysteroid UDP-glucosyltransferase (egt), and gp37). In this study, spectrofluorometry and western blot showed reduction of EGFP protein for all recombinant viruses with SV40 polyA, whereas qPCR showed an increase in the egfp mRNA levels. Therefore, we conclude that SV40 polyA increases mRNA levels but decreases protein production in the BEVS when the polyhedrin promoter is used at different loci. This work suggests that SV40 polyA in BEVSs should be replaced by an AcMNPV late gene polyA for optimal protein production or left untouched for optimal RNA production (RNA interference applications).     

Quantitative analysis of luciferase activity of viral and hybrid promoters in bovine preimplantation embryos

This study was conducted to investigate quantitatively the luciferase activity of gene constructs with viral and hybrid enhancers and promoters in bovine preimplantation embryos by using firefly luciferase reporter genes. In Experiment I, to examine the stability of the luciferase, bioluminescence intensity of bovine embryos injected with the luciferase gene driven by the SV40 early promoter and enhancer (SVEluc) was measured with a luminometer at 2 days after microinjection. The results indicated that the bioluminescence could be analysed at any time within 30 min because the luciferase activity was constant during the measurement period from 5 to 30 min. In Experiment II, the luciferase expression of fertilized oocytes injected with four gene constructs (TKEluc, TK6WEluc, SVEluc, and Miwluc) was analysed by using a photon imaging system at 2 or 6 days following microinjection. The results from Experiment II indicated that the reporter gene governed by the Miw promoter (RSV LTR and chicken β-actin promoter) was expressed more intensively in bovine morulae and blastocysts than three other gene constructs. In Experiment III, the effect of SV40 enhancer was investigated when fused downstream to the luciferase cDNA of the Miwluc vector. The results showed that SV40 enhancer further activated the luciferase activity of the Miw promoter in bovine preimplantation embryos. It was concluded, therefore, that the Miw promoter together with the SV40 enhancer would confer the strongest expression of the firefly luciferase reporter gene among the gene constructs tested in preimplantation bovine embryos. Mol. Reprod. Dev. 49:368–373, 1998. © 1998 Wiley-Liss, Inc.     

Expression of c-fos and c-myc protooncogenes in an immortalized hepatocyte line harbouring SV40 T antigen and hGH as transgenes

A clonal hepatocyte line (FMH-202-2), derived from livers of fetal transgenic mice harbouring human growth hormone (hGH) and SV40 T antigen as transgenes, was used in the investigation of protooncogene expression involved in liver-specific growth control and/or in hepatocellular transformation. In this model system, representing an immortalized, yet untransformed phenotype, the transgenes hGH and SV40 T antigen were expressed constitutively. The c-fos protooncogene was induced by incubation with insulin, epidermal growth factor (EGF) and insulin-like growth factor (IGF-I) in a transient manner comparable to its expression in primary murine hepatocytes. Elucidation of second messenger mechanisms demonstrated that c-fos induction by hepatotrophic growth factors was not mediated by protein kinase C. In contrast to primary hepatocytes, the c-myc protooncogene exhibited a constitutive expression pattern which was independent of growth factor stimulation. These results indicate that apart from hGH and SV40 T antigen, c-myc may play a role in cellular immortalization, but that constitutive expression of these genes, even in combined coexpression, does not suffice to induce the transformed phenotype.     

Immortalization of hypothalamic GnRH neurons by genetically targeted tumorigenesis

By genetically targeting tumorigenesis to specific hypothalamic neurons in transgenic mice using the promoter region of the gonadotropin-releasing hormone (GnRH) gene to express the SV40 T-antigen oncogene, we have produced neuronal tumors and developed clonal, differentiated, neurosecretory cell lines. These cells extend neurites, express the endogenous mouse GnRH mRNA, release GnRH in response to depolarization, have regulatable fast Na+ channels found in neurons, and express neuronal, but not glial, cell markers. These immortalized cells will provide an invaluable model system for study of hypothalamic neurosecretory neurons that regulate reproduction. Significantly, their derivation demonstrates the feasibility of immortalizing differentiated neurons by targeting tumorigenesis in transgenic mice to specific neurons of the CNS.     

Activation of PyMT in β Cells Induces Irreversible Hyperplasia,but Oncogene-Dependent Acinar Cell Carcinomas When Activated in Pancreatic Progenitors

It is unclear whether the cellular origin of various forms of pancreatic cancer involves transformation or transdifferentiation of different target cells or whether tumors arise from common precursors, with tumor types determined by the specific genetic alterations. Previous studies suggested that pancreatic ductal carcinomas might be induced by polyoma middle T antigen (PyMT) expressed in non-ductal cells. To ask whether PyMT transforms and transdifferentiates endocrine cells toward exocrine tumor phenotypes, we generated transgenic mice that carry tetracycline-inducible PyMT and a linked luciferase reporter. Induction of PyMT in β cells causes β-cell hyperplastic lesions that do not progress to malignant neoplasms. When PyMT is de-induced, β cell proliferation and growth cease; however, regression does not occur, suggesting that continued production of PyMT is not required to maintain the viable expanded β cell population. In contrast, induction of PyMT in early pancreatic progenitor cells under the control of Pdx1 produces acinar cell carcinomas and β-cell hyperplasia. The survival of acinar tumor cells is dependent on continued expression of PyMT. Our findings indicate that PyMT can induce exocrine tumors from pancreatic progenitor cells, but cells in the β cell lineage are not transdifferentiated toward exocrine cell types by PyMT; instead, they undergo oncogene-dependent hyperplastic growth, but do not require PyMT for survival.     

Impaired CK1 delta activity attenuates SV40-induced cellular transformation in vitro and mouse mammary carcinogenesis in vivo

Simian virus 40 (SV40) is a powerful tool to study cellular transformation in vitro, as well as tumor development and progression in vivo. Various cellular kinases, among them members of the CK1 family, play an important role in modulating the transforming activity of SV40, including the transforming activity of T-Ag, the major transforming protein of SV40, itself. Here we characterized the effects of mutant CK1δ variants with impaired kinase activity on SV40-induced cell transformation in vitro, and on SV40-induced mammary carcinogenesis in vivo in a transgenic/bi-transgenic mouse model. CK1δ mutants exhibited a reduced kinase activity compared to wtCK1δ in in vitro kinase assays. Molecular modeling studies suggested that mutation N172D, located within the substrate binding region, is mainly responsible for impaired mutCK1δ activity. When stably over-expressed in maximal transformed SV-52 cells, CK1δ mutants induced reversion to a minimal transformed phenotype by dominant-negative interference with endogenous wtCK1δ. To characterize the effects of CK1δ on SV40-induced mammary carcinogenesis, we generated transgenic mice expressing mutant CK1δ under the control of the whey acidic protein (WAP) gene promoter, and crossed them with SV40 transgenic WAP-T-antigen (WAP-T) mice. Both WAP-T mice as well as WAP-mutCK1δ/WAP-T bi-transgenic mice developed breast cancer. However, tumor incidence was lower and life span was significantly longer in WAP-mutCK1δ/WAP-T bi-transgenic animals. The reduced CK1δ activity did not affect early lesion formation during tumorigenesis, suggesting that impaired CK1δ activity reduces the probability for outgrowth of in situ carcinomas to invasive carcinomas. The different tumorigenic potential of SV40 in WAP-T and WAP-mutCK1δ/WAP-T tumors was also reflected by a significantly different expression of various genes known to be involved in tumor progression, specifically of those involved in wnt-signaling and DNA repair. Our data show that inactivating mutations in CK1δ impair SV40-induced cellular transformation in vitro and mouse mammary carcinogenesis in vivo.     

A Transgenic Mouse Model with Inducible Tyrosinase Gene Expression Using the Tetracycline (Tet‐on) System Allows Regulated Rescue of Abnormal Chiasmatic Projections Found in Albinism

Congenital defects in retinal pigmentation, as in oculocutaneous albinism Type I (OCA1), where tyrosinase is defective, result in visual abnormalities affecting the retina and pathways into the brain. Transgenic animals expressing a functional tyrosinase gene on an albino genetic background display a correction of all these abnormalities, implicating a functional role for tyrosinase in normal retinal development. To address the function of tyrosinase in the development of the mammalian visual system, we have generated a transgenic mouse model with inducible expression of the tyrosinase gene using the tetracycline (TET‐ON) system. We have produced two types of transgenic mice: first, mice expressing the transactivator rtTA chimeric protein under the control of mouse tyrosinase promoter and its locus control region (LCR), and; second, transgenic mice expressing a mouse tyrosinase cDNA construct driven by a minimal promoter inducible by rtTA in the presence of doxycycline. Inducible experiments have been carried out with selected double transgenic mouse lines. Tyrosinase expression has been induced from early embryo development and its impact assessed with histological and biochemical methods in heterozygous and homozygous double transgenic individuals. We have found an increase of tyrosinase activity in the eyes of induced animals, compared with littermate controls. However, there was significant variability in the activation of this gene, as reported in analogous experiments. In spite of this, we could observe corrected uncrossed chiasmatic pathways, decreased in albinism, in animals induced from their first gestational week. These mice could be instrumental in revealing the role of tyrosinase in mammalian visual development.     

Transgenic mouse models for tumors of melanocytes and retinal pigment epithelium

Cutaneous and ocular melanomas are due to malignant transformation of neural crest-derived melanocytes. The rising incidence of this tumor in humans has stimulated experiments to devise suitable mouse models. In the past years, transgenic mouse lines have been generated using different oncogenes - Ha-ras, SV40 T antigen (Tag), ret - which develop benign lesions of melanocytes, melanoma, and/or eye tumors. Pigment cell tumors in humans, although rather rare, can also develop from the retinal pigment epithelium (RPE), a cell layer of neuroectodermal origin. We, therefore, established transgenic models for this ocular tumor. Regulated by the promoter of tyrosinase-related protein-1 (TRP-1), two oncogenes, ret and SV40 Tag, were targeted to the developing RPE in transgenic mice. The TRP-1/ret transgenic mice displayed microphthalmia and benign tumors of the RPE. Expression of SV40 T antigen (TRP-1/Tag) led to malignant tumors, which were invasive and metastasized to inguinal lymph node and spleen.