Hyperplasia and impaired involution in the mammary gland of transgenic mice expressing human FGF4

Fgf4, a member of the fibroblast growth factor family, is frequently amplified in a variety of human cancers, however, its expression in neoplastic tissues is rarely detectable. This makes uncertain its involvement in tumour aetiology, although several in-vitro studies link Fgf4 overexpression to malignant transformation and metastatization of culture cells. We generated a transgenic mouse model in which the whey acidic protein (WAP) promoter directs expression of human Fgf4 to mammary tissues during late pregnancy and throughout lactation, with the purpose of studying the involvement of this growth factor in mammary tumorigenesis. Expression of the transgene was specifically detected in lobular-alveolar cells of lactating mammary glands that, by histological analysis, displayed hyperplastic areas and a disorganized structure. This was accompanied by an increased number of red blood cells and expression, in alveolar epithelial cells, of the vascular endothelial growth factor, which is absent in wild type controls. The most striking effect caused by FGF4 overexpression was on the remodelling of mammary tissue at the end of lactation. Indeed, transgenic animals showed a delayed involution of the gland due to a dramatic reduction in the overall number of apoptotic cells, which are normally present in the organ after weaning. Nevertheless, none of the animals examined developed neoplastic lesions of the mammary gland even after several pregnancies and at old age. Our work represents the first in-vivo demonstration of the anti-apoptotic and angiogenic properties of FGF4.     

Apoptosis and tumor resistance conferred by Par-4

Par-4 is a tumor suppressor protein with a pro-apoptotic function. Epigenetic silencing of Par-4 is seen in diverse tumors and Par-4 knockout mice develop spontaneous tumors in various tissues. Endogenous Par-4 is essential for sensitization of cells to diverse apoptotic stimuli, whereas ectopic expression of Par-4 can selectively induce apoptosis in cancer cells. The cancer-specific pro-apoptotic action of Par-4 resides in its centrally located SAC domain. This review emphasizes the role of Par-4/SAC in apoptosis and tumor resistance. SAC transgenic mice display normal development and life span, and, most importantly, are resistant to spontaneous, as well as oncogene-induced, autochthonous tumors. The tumor resistant phenotype and undetectable toxicity of SAC in vivo suggests the SAC domain possesses tremendous therapeutic potential.     

Hepatomegaly in transgenic mice expressing an oncogenic form of beta-catenin

Inappropriate activation of the Wnt/beta-catenin signaling, resulting mainly from activating mutations of the beta-catenin gene, has been implicated recently in the development of hepatocellular carcinoma (HCC). We have generated transgenic mice expressing an oncogenic form of beta-catenin in their hepatocytes to analyze the effect of deregulated beta-catenin signaling on liver homeostasis. These mice rapidly developed hepatomegaly soon after birth, with livers three to four times heavier than those of nontransgenic littermates. The liver cell hyperplasia resulted from increased cell proliferation without any compensatory apoptosis. Although the genes encoding c-myc and cyclin D1 are potential targets of the beta-catenin signaling pathway, neither of them was overexpressed in the hyperplastic livers of beta-catenin transgenic mice. Thus, the key target genes of the beta-catenin signaling pathway in the liver remain to be identified.     

Mammary tumor induction in transgenic mice expressing an RNA-binding protein

We have analyzed mammary tumors arising in transgenic mice expressing a novel, multifunctional RNA-binding protein. The protein, which we call the c-myc mRNA coding region instability determinant binding protein (CRD-BP), binds to c-myc, insulin-like growth factor II, and beta-actin mRNAs, and to H19 RNA. Depending on the RNA substrate, the CRD-BP affects RNA localization, translation, or stability. CRD-BP levels are high during fetal development but low or undetectable in normal adult tissues. The CRD-BP is linked to tumorigenesis, because its expression is reactivated in some adult human breast, colon, and lung tumors. These data suggest the CRD-BP is a proto-oncogene. To test this idea, the CRD-BP was expressed from the whey acidic protein (WAP) promoter in mammary epithelial cells of adult transgenic mice. The incidence of mammary tumors was 95% and 60% in two lines of WAP-CRD-BP mice with high and low relative CRD-BP expression, respectively. Some of the tumors metastasized. Nontransgenic mice did not develop mammary tumors. H19 RNA and insulin-like growth factor II mRNA were up-regulated significantly in non-neoplastic WAP-CRD-BP mammary tissue. WAP-CRD-BP mice are a novel model for mammary neoplasia and might provide insights into human breast cancer biology.     

Myeloid leukemia with promyelocytic features in transgenic mice expressing hCG-NuMA-RARalpha

Acute promyelocytic leukemia (APL) is characterized by the accumulation of abnormal promyelocytes in the bone marrow (BM), and by the presence of a reciprocal chromosomal translocation involving retinoic acid receptor alpha (RARalpha). To date, five RARalpha partner genes have been identified in APL. NuMA-RARalpha was identified in a pediatric case of APL carrying a translocation t(11;17)(q13;q21). Using a construct containing the NuMA-RARalpha fusion gene driven by the human cathepsin G promoter (hCG-NuMA-RARalpha), two transgenic mouse lines were generated. Transgenic mice were observed to have a genetic myeloproliferation (increased granulopoiesis in BM) at an early age, and rapidly developed a myeloproliferative disease-like myeloid leukemia. This leukemia was morphologically and immunophenotypically indistinguishable from human APL, with a penetrance of 100%. The phenotype of transgenic mice was consistent with a blockade of neutrophil differentiation. NuMA-RARalpha is therefore sufficient for disease development in this APL model.     

Tumorigenesis and eye abnormalities in transgenic mice expressing MSV-SV40 large T-antigen

Transgenic mice which expressed SV40 large T-antigen under the control of the MSV enhancer and the SV40 promoter were generated. In animals containing an intact MSV enhancer, total lens cataracts and neuroectodermal brain tumors, originating in the pineal organ were observed. In contrast, 5' deletion of the MSV enhancer to a residual 53 bp resulted in a different spectrum of pathologies. Whilst lens cataracts still occurred, no brain tumors could be detected. Instead, fibrosarcomas and adenocarcinomas of the kidneys were induced. In addition, tumors of the endocrine pancreas were observed with both transgene constructs. We conclude that the MSV enhancer element is sufficient to direct the expression of the viral reporter gene to the lens and the pineal organ in transgenic mice. Deletion of the MSV enhancer correlates with the loss of DNA elements responsible for the pineal cell specific expression of SV40 large T-antigen.     

Black cohosh increases metastatic mammary cancer in transgenic mice expressing c-erbB2

Black cohosh is an herbal extract that is often used as an alternative to estrogen-based replacement therapies to treat hot flushes that frequently accompany the transition to menopause. Although cancer-free women as well as breast cancer patients and survivors use black cohosh to relieve vasomotor symptoms, there is limited information on its potential to influence breast cancer development or progression. Therefore, in this study, the effects of black cohosh on mammary tumorigenesis were investigated in the MMTV-neu mouse model due to its similarities to HER2(+) breast cancer, including stochastic development of mammary tumors, which frequently progress to metastatic disease. Using an adjusted dose for the mice to correlate to the recommended dose in women (40 mg/d), no differences were detected in the incidence or onset of mammary tumors in black cohosh-treated versus control females. The lack of effect on mammary tumor development suggests that black cohosh would not influence breast cancer risk if given to women before tumor formation. In contrast, black cohosh significantly increased the incidence of lung metastases in tumor-bearing animals compared with mice fed the isoflavone-free control diet. Additional studies will be needed to correlate these findings to women taking different black cohosh products at various times during breast cancer development; however, these results suggest caution for women using black cohosh, especially for extended periods of time. As metastatic progression is linked to patient survival, these data stress the importance of investigating how women's therapies influence all stages of mammary tumorigenesis, particularly for assessing their safety.     

Reduced mammary gland carcinogenesis in transgenic mice expressing a growth hormone antagonist

Several reports have provided evidence that body size early in life is positively correlated with risk of subsequent breast cancer, but the biological basis for this relationship is unclear. We examined tumour incidence in transgenic mice expressing a growth hormone (GH) antagonist and in non-transgenic littermates following exposure to dimethylbenz[ a ]anthracene (DMBA), a well characterized murine mammary gland carcinogen. The transgenic animals had lower IGF-I levels, were smaller in terms of body size and weight, and exhibited decreased tumour incidence relative to controls. The demonstration that both body size early in life and breast cancer incidence are influenced by experimental perturbation of the GH-IGF-I axis in a transgenic model provides evidence that variability between individuals with respect to these hormones underlies the relationship between body size early in life and breast cancer risk observed in epidemiological studies.     

Alterations of the thymic selection process in transgenic mice expressing SV40 large T antigen

We generated SV40 T antigen transgenic mice (lines SVT125, SVT127, and SVT248) which developed unique thymic carcinomas originating from thymic cortical epithelial cells. In these mice we observed alterations in the thymic selection process not reported before in SV40 T antigen transgenic mice. Along with tumor cell growth, thymocytes increased in number and the proportion of CD4 or CD8 single positive cells rose to 10 times the normal level. Expression of SV40 T antigen was detectable by Northern analysis in thymic stromal cells but not in thymocytes. Thymic stromal cell lines, derived from the thymic tumor, produced high levels of cytokines which caused morphological transformation and growth stimulation in hematopoietic stem cells, including fetal liver cells and bone marrow cells. These observations suggest that the unusual multiplication of thymocytes and the alterations in thymic selection are the result of the activity of thymic stromal cells transformed by SV40 T antigen. The cell lines derived from the tumor can thus be used to study cytokines involved in thymic differentiation of T cells. © 1996 Wiley-Liss, Inc.     

Mammary cancer in transgenic mice expressing insulin-like growth factor II (IGF-II)

The effect of insulin-like growth factor II (IGF-II) on tumour development in the mouse mammary gland was studied. To promote extra IGF-II expression in the mammary gland, sheep beta-lactoglobulin regulatory elements were attached to the coding regions of the mouse Igf-2 gene and injected into the pronuclei of mouse zygotes. Mammary tumours developed in each of the four independent lines of mice which expressed transgene IGF-II in the gland. Tumours from two of the lines grew after transplantation to both male and female hosts. Primary tumours contained stromal and epithelial regions, but the tumours were dominated by mammary adenocarcinoma after transplantation. The tumours expressed high levels of Igf-2 mRNA transcribed from the integrated transgenes.     

Autonomous growth and hepatocarcinogenesis in transgenic mice expressing the p53 family inhibitor DNp73

p53 family proteins carry on a wide spectrum of biological functions from differentiation, cell cycle arrest, apoptosis and chemosensitivity of tumors. Conversely, N-terminally truncated p73 (DNp73) functions as a potent inhibitor of all these tumor suppressor properties, implicating its participation in malignant transformation and oncogenesis. Several reports indicated considerable up-regulation of DNp73 in hepatocellular carcinoma (HCC) that correlates with reduced survival of patients, but little is known about the functional significance of DNp73 to tumorigenesis in vivo due to the lack of an appropriate model. To address this, we generated transgenic mice in which DNp73 expression is directed to the liver by the albumin promoter. Gene expression was tested by mRNA and protein analyses. Transgenic mice exhibited prominent hepatic histological abnormalities including increased hepatocyte proliferation resulting in preneoplastic lesions (liver cell adenomas) at 3-4 months. Among 12- to 20-month-old mice, 83% of animals developed hepatic carcinoma. HCC displayed a significant increase of hyperphosphorylated inactive retinoblastoma, whereas p53-regulated inhibitors of cell cycle progression were down-regulated in the tumors. Our data firmly establish the unique oncogenic capability of DNp73 to drive hepatocarcinogenesis in vivo, supporting its significance as a marker for disease severity in patients and as target for cancer prevention. This model offers new opportunities to further delineate DNp73-mediated liver oncogenesis but may also enable the development of more effective cancer therapies.     

Enhanced malignant tumorigenesis in Cdk4 transgenic mice

In a previous study, we reported that overexpression of cyclin-dependent kinase-4 (CDK4) in mouse epidermis results in epidermal hyperplasia, hypertrophy and severe dermal fibrosis. In this study, we have investigated the susceptibility to skin tumor formation by forced expression of CDK4. Skin tumors from transgenic mice showed a dramatic increase in the rate of malignant progression to squamous cell carcinomas (SCC) in an initiation-promotion protocol. Histopathological analysis of papillomas from transgenic mice showed an elevated number of premalignant lesions characterized by dysplasia and marked atypia. Interestingly, transgenic mice also developed tumors in initiated but not promoted skin, demonstrating that CDK4 replaced the action of tumor promoters. These results suggest that expression of cyclin D1 upon ras activation synergizes with CDK4 overexpression. However, cyclin D1 transgenic mice and double transgenic mice for cyclin D1 and CDK4 did not show increased malignant progression in comparison to CDK4 transgenic mice. Biochemical analysis of tumors showed that CDK4 sequesters the CDK2 inhibitors p27Kip1 and p21Cip1, suggesting that indirect activation of CDK2 plays an important role in tumor development. These results indicate that, contrary to the general assumption, the catalytic subunit, CDK4, has higher oncogenic activity than cyclin D1, revealing a potential use of CDK4 as therapeutic target.     

Differential expression levels of Par-4 in melanoma

The pro-apoptotic prostate apoptosis response-4 gene product Par-4 sensitizes prostate cells to the induction of programmed cell death. In this study we examined Par-4 expression in human melanoma cell lines and melanoma metastases. The heterogeneous expression detected prompted us to investigate the biological relevance of Par-4 in a human melanoma xenotransplantation model. Overexpression of Par-4 by transfection decreased tumour development in xenotransplanted A375-C6 melanoma cells in SCID mice and correlated to an increase in tumour cell apoptosis. These data suggest that high expression of the pro-apoptotic protein Par-4 could qualify as a prognostic marker in human melanoma.     

Early development of polycystic kidney disease in transgenic mice expressing an activated mutant of the beta-catenin gene

Autosomal dominant polycystic kidney disease (ADPKD) is common and is a major cause of renal failure. Although the genetics of ADPKD are well known and have led to the discovery of polycystins, a new protein family, the pathogenesis of the disease remains largely unknown. Recent studies have indicated that the beta-catenin signaling pathway is one of the targets of the transduction pathway controlled by the polycystins. We have generated transgenic mice that overproduce an oncogenic form of beta-catenin in the epithelial cells of the kidney. These mice developed severe polycystic lesions soon after birth that affected the glomeruli, proximal, distal tubules and collecting ducts. The phenotype of these mice mimicked the human ADPKD phenotype. Cyst formation was associated with an increase in cell proliferation and apoptosis. The cell proliferation and apoptotic indexes was increased 4-5-fold and 3-4-fold, respectively, in cystic tubules of the transgenic mice compared to that of littermate controls. Our findings provide experimental genetic evidence that activation of the Wnt/beta-catenin signaling pathway causes polycystic kidney disease and support the view that dysregulation of the Wnt/beta-catenin signaling is involved in its pathogenesis.     

Enhanced repair of O6-methylguanine DNA adducts in the liver of transgenic mice expressing the ada gene

The capacity to repair O6-methylguanine-DNA adducts was measured in the liver of transgenic mice expressing a chimeric gene consisting of the inducible P-enolpyruvate carboxykinase (GTP) promoter linked to the bacterial O6-alkylguanine-DNA alkyltransferase (ada) gene. Under induced conditions, total hepatic alkyltransferase reached 32.8 +/- 4.2 (SE) fmol/micrograms DNA compared to 7.8 +/- 1.1 fmol/micrograms DNA in nontransgenic mice. Administration of methylnitrosourea or nitrosodimethylamine to both groups of mice produced O6-methylguanine-DNA adducts which resulted in repair-mediated depletion of total hepatic alkyltransferase in a dose-dependent fashion. In nontransgenic mice, depletion of hepatic alkyltransferase occurred at lower doses of carcinogen, and recovery of alkyltransferase activity occurred later than in ada+ transgenic mice. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of residual alkyltransferase activity after methylating agent exposure indicated that the bacterial as well as endogenous mammalian alkyltransferases were functioning as DNA repair proteins in hepatocytes in vivo. Analysis of O6-methylguanine- and N7-methylguanine-DNA adducts in the liver of transgenic and nontransgenic mice after treatment with one dose of 50 mg/kg methylnitrosourea i.p. revealed that transgenic mice repaired in situ O6-methylguanine-DNA adducts approximately 3 times faster than nontransgenic mice, commensurate with the increase in alkyltransferase activity. Thus, ada+ transgenic mice treated with methylnitrosourea have lower levels of persistent mutagenic O6-methylguanine adducts than ada- nontransgenic mice. Hepatic expression of bacterial alkyltransferase appears to protect mice from the DNA-damaging effects of N-nitroso compounds in vivo.     

Histopathology of salivary and mammary gland tumors in transgenic mice expressing a human Ha-ras oncogene

Mutated ras genes are powerful transforming agents in vitro and are found in a wide variety of human tumors in vivo. We characterized the histopathology and p21 protein expression associated with tumorigenesis in line 69 transgenic mice carrying an activated, human c-Ha-ras gene on the Y-chromosome (A. C. Andres, C. A. Schonenberger, B. Groner, L. Hennighausen, M. LeMeur, and P. Gerlinger, Proc. Natl. Acad. Sci. USA, 84: 1299-1303, 1987). Male mice developed salivary and/or mammary gland tumors. The salivary tumors were adenosquamous carcinomas arising from serous areas of the submandibular gland. They characteristically exhibited densely packed cords and sheets of moderately anaplastic cells. Tumorigenic tissue had a high mitotic index, and all tumor-bearing animals had an ongoing inflammatory response as evidenced by extensive immune cell infiltration of affected tissue. Half of the mammary gland tumors were adenosquamous carcinomas with multiple foci of squamous metaplasia, while the rest were adenocarcinomas containing glandular tissue. Most tumors had a high mitotic index, and abnormal mitotic figures were common. All tumors produced p21 ras, as confirmed by immunohistochemistry and Western blots. Both tumor types expressed elevated levels of p21 protein. Microscopic lung metastases were present in 5 of 35 animals (14%). Our results suggest that this transgenic mouse will provide a useful model for testing therapies directed against ras-associated tumorigenesis.