The Rgr oncogene induces tumorigenesis in transgenic mice

To study the oncogenic potential of Rgr in vivo, we have generated several transgenic Rgr mouse lines, which express the oncogene under the control of different promoters. These studies revealed that Rgr expression leads to the generation of various pathological alterations, including fibrosarcomas, when its transgenic expression is restricted to nonlymphoid tissues. Moreover, the overall incidence and latency of fibrosarcomas were substantially increased and shortened, respectively, in a p15INK4b-defective background. More importantly, we also have demonstrated that Rgr expression in thymocytes of transgenic mice induces severe alterations in the development of the thymocytes, which eventually lead to a high incidence of thymic lymphomas. This study demonstrates that oncogenic Rgr can induce expression of p15INK4b and, more importantly, that both Rgr and p15INK4b cooperate in the malignant phenotype in vivo. These findings provide new insights into the tumorigenic role of Rgr as a potent oncogene and show that p15INK4b can act as a tumor suppressor gene.     

Overexpression of Id1 in transgenic mice promotes mammary basal stem cell activity and breast tumorigenesis

Inhibitor of differentiation/DNA binding (Id)1 is a crucial regulator of mammary development and breast cancer progression. However, its effect on stemness and tumorigenesis in mammary epithelial cells remains undefined. Herein, we demonstrate that Id1 induces mammary tumorigenesis by increasing normal and malignant mammary stem cell (MaSC) activities in transgenic mice. MaSC-enriched basal cell expansion and increased self-renewal and in vivo regenerative capacity of MaSCs are observed in the mammary glands of MMTV-Id1 transgenic mice. Furthermore, MMTV-Id1 mice develop ductal hyperplasia and mammary tumors with highly expressed basal markers. Id1 also increases breast cancer stem cell (CSC) population and activity in human breast cancer lines. Moreover, the effects of Id1 on normal and malignant stem cell activities are mediated by the Wnt/c-Myc pathway. Collectively, these findings provide in vivo genetic evidence of Id1 functions as an oncogene in breast cancer and indicate that Id1 regulates mammary basal stem cells by activating the Wnt/c-Myc pathway, thereby contributing to breast tumor development.     

Human Kaposi's sarcoma cell-mediated tumorigenesis in human immunodeficiency type 1 tat-expressing transgenic mice

BACKGROUND: The human immunodeficiency virus type 1 (HIV-1) transactivator (Tat) protein has been linked to the development and course of Kaposi's sarcoma (KS) associated with acquired immunodeficiency disease syndrome (AIDS-KS). Tat is an 86-101 amino-acid protein encoded by two exons. To evaluate the growth-promoting effects of Tat in AIDS-KS in vivo, we developed transgenic mice expressing the one-exon-encoded 72 amino-acid protein (Tat(72)) and the two-exon-encoded 86 amino-acid protein (Tat(86)). METHODS: Human KS SLK cells were injected subcutaneously into CD4(+) T-cell-depleted male mice, and the tumors that formed after 3-4 weeks were recovered and analyzed for the expression of Tat protein(s), different cytokine messenger RNAs (mRNAs), and matrix metalloproteinases (MMPs). All statistical tests were two-sided. RESULTS: The average tumor weight was maximum in Tat(86) mice ( approximately 600 mg) compared with Tat(72) ( approximately 200 mg) and nontransgenic ( approximately 100 mg) mice (P<.005). Histologic examination of tumors showed spindle-shaped SLK cells with prominent infiltrates of inflammatory cells. All of the tumors from Tat mice expressed abundant Tat mRNA, suggesting that the infiltrating mouse cells actively expressed Tat. A comparison of the growth-promoting cytokines in the tumors from Tat(86)-transgenic and nontransgenic mice showed that the expression of the following cytokines was substantially increased in the tumors of the Tat(86) mice: tumor necrosis factor-alpha, interleukin 6, interleukin 8, granulocyte-macrophage colony-stimulating factor, and basic fibroblast growth factor. Furthermore, these tumors showed abundant expression of a 105-kd MMP activity associated with infiltrates of host leukocytes in the lesions. CONCLUSION: Our in vivo data clearly suggest that extracellular Tat can contribute to the growth and tumorigenesis of human KS cells.     

Overexpression of NPM-ALK induces different types of malignant lymphomas in IL-9 transgenic mice

Anaplastic large-cell lymphoma (ALCL) comprises approximately 25% of all non-Hodgkin lymphomas (NHL) in children and young adults, and up to 15% of high-grade NHL in older patients. Over 50% of these tumours carry the translocation t(2;5)(p23;q35). The result of this translocation is the fusion of the nucleophosmin (NPM) gene to the anaplastic lymphoma kinase (ALK) gene. The resulting hybrid protein contains the ALK catalytic domain that consequently confers transforming potential, which contributes to the pathogenesis of ALCL. To further analyse the transforming activity in an animal model, a cDNA encoding the protein product, NPM-ALK, was inserted into the retrovirus vector pLXSN and transduced into mouse bone marrow progenitors. These cells were subsequently used in a bone marrow transplant with the aim of reconstituting the haematopoietic compartments of lethally irradiated recipients. IL-9 transgenic mice were chosen as the animal model system, because dysregulated expression of the IL-9 gene in transgenic mice results in the sporadic development of spontaneous thymic lymphomas. Moreover, IL-9 is known to be expressed in cases of human ALCL. We used 15 IL-9 transgenic mice and eight corresponding wild-type mice (FVB/N) and transplanted them with NPM/ALK infected bone marrow cells. Eight IL-9 transgenic mice, serving as a control group, received pLXSN (vector only)-infected marrow. Reconstituted mice developed NPM-ALK-positive lymphomas, including lymphoblastic lymphomas of T-cell type (T-LB), mature and immature plasmacytoma (PC), and plasmoblastic/anaplastic diffuse large-B-cell lymphoma after about 19-20 weeks. The combined overexpression of NPM-ALK and IL-9 led to the transformation of murine lymphoid cells with accelerated and enhanced development of T-LB in 46% of the mice, which only very rarely occurs in IL-9 transgenic mice only. Of the 15 animals, five (33%) developed plasmacytic/plasmoblastic neoplasms, of which the most aggressive tumours share many features with anaplastic/plasmoblastic diffuse large-B-cell lymphoma on the basis of morphology, a characteristic growth pattern and ALK expression.     

Overexpression of interleukin-1beta induces gastric inflammation and cancer and mobilizes myeloid-derived suppressor cells in mice

Polymorphisms of interleukin-1beta (IL-1beta) are associated with an increased risk of solid malignancies. Here, we show that stomach-specific expression of human IL-1beta in transgenic mice leads to spontaneous gastric inflammation and cancer that correlate with early recruitment of myeloid-derived suppressor cells (MDSCs) to the stomach. IL-1beta activates MDSCs in vitro and in vivo through an IL-1RI/NF-kappaB pathway. IL-1beta transgenic mice deficient in T and B lymphocytes develop gastric dysplasia accompanied by a marked increase in MDSCs in the stomach. Antagonism of IL-1 receptor signaling inhibits the development of gastric preneoplasia and suppresses MDSC mobilization. These results demonstrate that pathologic elevation of a single proinflammatory cytokine may be sufficient to induce neoplasia and provide a direct link between IL-1beta, MDSCs, and carcinogenesis.     

Asbestos induces activator protein-1 transactivation in transgenic mice

Activation of activator protein (AP-1) by crocidolite asbestos was examined in vitro in a JB6 P+ cell line stably transfected with AP-1-luciferase reporter plasmid and in vivo using AP-1-luciferase reporter transgenic mice. In in vitro studies, crocidolite asbestos caused a dose- and time-dependent induction of AP-1 activation in cultured JB6 cells. The elevated AP-1 activity persisted for at least 48 h. Crocidolite asbestos also induced AP-1 transactivation in the pulmonary and bronchial tissues of transgenic mice. AP-1 activation was observed at 2 days after intratracheal instillation of the mice with asbestos. At 3 days postexposure, AP-1 activation was elevated 10-fold in the lung tissue and 22-fold in bronchiolar tissue as compared with their controls. The induction of AP-1 activity by asbestos appeared to be mediated through the activation of mitogen-activated protein kinase family members, including extracellular signal-regulating protein kinase, Erk1 and Erk2. Aspirin inhibited asbestos-induced AP-1 activity in JB6 cells. Pretreatment of the mice with aspirin also inhibited asbestos-induced AP-1 activation in bronchiolar tissue. The data suggest that further investigation of the role of AP-1 activation in asbestos-induced cell proliferation and carcinogenesis is warranted. In addition, investigation of the potential therapeutic benefits of aspirin in the prevention/amelioration of asbestos-induced cancer is justified.     

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.     

HMGA2 induces pituitary tumorigenesis by enhancing E2F1 activity

HMGA2 gene amplification and overexpression in human prolactinomas and the development of pituitary adenomas in HMGA2 transgenic mice showed that HMGA2 plays a crucial role in pituitary tumorigenesis. We have explored the pRB/E2F1 pathway to investigate the mechanism by which HMGA2 acts. Here we show that HMGA2 interacts with pRB and induces E2F1 activity in mouse pituitary adenomas by displacing HDAC1 from the pRB/E2F1 complex-a process that results in E2F1 acetylation. We found that loss of E2F1 function (obtained by mating HMGA2 and E2F1(-/-) mice) suppressed pituitary tumorigenesis in HMGA2 mice. Thus, HMGA2-mediated E2F1 activation is a crucial event in the onset of these tumors in transgenic mice and probably also in human prolactinomas.     

Parity-induced mammary epithelial cells facilitate tumorigenesis in MMTV-neu transgenic mice

Using a Cre-lox-based genetic labeling technique, we have recently discovered a parity-induced mammary epithelial subtype that is abundant in nonlactating and nonpregnant, parous females. These mammary epithelial cells serve as alveolar progenitors in subsequent pregnancies, and transplantation studies revealed that they possess features of multipotent progenitors such as self-renewal and the capability to contribute to ductal and alveolar morphogenesis. Here, we report that these cells are the cellular targets for transformation in MMTV-neu transgenic mice that exhibit accelerated mammary tumorigenesis in multiparous animals. The selective ablation of this epithelial subtype reduces the onset of tumorigenesis in multiparous MMTV-neu transgenics. There is, however, experimental evidence to suggest that parity-induced mammary epithelial cells may not be the only cellular targets in other MMTV-promoter-based transgenic strains. In particular, the heterogeneous MMTV-wnt1 lesions predominantly express the ductal differentiation marker Nkcc1 that is absent in MMTV-neu-derived tumors. Our observations support the idea that tumors originate from distinctly different epithelial subtypes in selected MMTV-promoter-driven cancer models and that diverse oncogenes might exert discrete effects on particular mammary epithelial subtypes.     

Overexpression of 15-lipoxygenase-1 in PC-3 human prostate cancer cells increases tumorigenesis.

The effect of overexpression of 15-lipoxygenase-1 (15-LO-1) was studied in the human prostate cancer cell line, PC-3. Stable PC-3 cell lines were generated by transfection with 15-LO-1-sense (15-LOS), 15-LO-1-antisense (15-LOAS) or vector (Zeo) and selection with Zeocin. After characterization by RT-PCR, western and HPLC, a PC3-15LOS clone was selected that possessed 10-fold 15-LO-1 enzyme activity compared with parental PC-3 cells. The PC3-15LOAS clone displayed little or no 15-LO-1 activity. These PC-3 cell lines were characterized for properties of tumorigenesis. The proliferation rates of the cell lines were as follows: PC3-15LOS > PC-3 = PC3-Zeo > PC3-15LOAS. Addition of a specific 15-LO-1 inhibitor, PD146176, caused a dose-dependent inhibition of proliferation in vitro. Overexpression of 15-LO-1 also caused [(3)H]thymidine incorporation to increase by 4.0-fold (P < 0.01). Compared with parental and PC-3-Zeo cells, PC3-15LOS enhanced whereas PC3-15LOAS reduced the ability of PC-3 cells to grow in an anchorage-independent manner, as assessed by colony formation in soft agar. These data suggested a pro-tumorigenic role for 15-LO-1 in PC-3 cells in vitro. Therefore, to clarify the role of 15-LO-1 in vivo, the effect of 15-LO-1 expression on the growth of tumors in nude mice was investigated. The PC-3 cell lines were inoculated subcutaneously into athymic nude mice. The frequency of tumor formation was increased and the sizes of the tumors formed were much larger in the PC3-15LOS compared with PC3-15LOAS, parental PC-3 and PC-3-Zeo cells. Immunohistochemistry for 15-LO-1 confirmed expression throughout the duration of the experiment. The expression of factor VIII, an angiogenesis marker, in tumor sections was increased in tumors derived from PC3-15LOS cells and decreased in those from PC3-15LOAS cells compared with tumors from parental or Zeo cells. These data further supported the evaluation by ELISA of vascular endothelial growth factor (VEGF) secretion by PC-3 cells in culture. Secretion of this angiogenic factor was elevated in PC3-15LOS cells compared with the other cell lines. These results support a role for 15-LO-1 in a novel growth-promoting pathway in the prostate.     

Conditional E2F1 activation in transgenic mice causes testicular atrophy and dysplasia mimicking human CIS

E2F1 is a crucial downstream effector of the retinoblastoma protein (pRB) pathway. To address the consequences of short-term increase in E2F1 activity in adult tissues, we generated transgenic mice expressing the human E2F1 protein fused to the oestrogen receptor (ER) ligand-binding domain. The expression of the ER-E2F1 fusion protein, which is inactive in the absence of 4-hydroxy tamoxifen (OHT), was targeted to the testes. We show that short-term activation of E2F1 results in activation of E2F target genes and apoptosis of germ cells. Consistent with our previously published results, the apoptotic response was independent of p53. Persistent E2F1 activation for 3 weeks led to massive apoptosis and severe testicular atrophy with seminiferous tubules containing only Sertoli cells and clusters of undifferentiated spermatogonia. The latter showed high expression of ER-E2F1 and excessive mitotic activity, including atypical mitoses. In addition, gonocyte-like dysplastic germ cells, resembling carcinoma in situ (CIS) cells in humans, appeared. Our results show that a relatively short period of deregulated E2F1 activity in testicles can induce premalignant changes. Moreover, we demonstrate the feasibility of tissue-specific expression of conditional ER-E2F1 in transgenic mice.     

c-fos expression induces bone tumors in transgenic mice

The proto-oncogene c-fos has been isolated as the cellular homolog of the v-fos gene found in the osteosarcoma inducing FBR- and FBJ-murine sarcoma viruses (MSV). Expression of the c-fos gene in transgenic mice leads to the development of bone lesions of which about half progress to bone tumors mainly chondrosarcomas. The tumors display a strong preference for males and have a latency with a mean of 9.5 months. However, also mice without visible lesions develop bone tumors with the same sex preference and latency. These consequences of c-fos expression are independent of the chosen promoter but dependent on a replacement of 3' noncoding sequences of c-fos by a long terminal repeat (LTR) of the FBJ-MSV virus.     

The role of human prolactin and its antagonist, G129R, in mammary gland development and DMBA-initiated tumorigenesis in transgenic mice

Human prolactin (hPRL) has been implicated to have a pathological role in breast cancer and play a critical role in mammary gland development. The hPRL antagonist, G129R, has been shown to induce breast cancer cell apoptosis. 9,10-Dimethyl-1,2-benzanthracene (DMBA), a potent mammary gland carcinogen, induces hormone responsive mammary tumor formation in rodents. To investigate the effects of hPRL and its counterpart, G129R, on mammary gland development and tumorigenesis, transgenic mice that express hPRL or G129R under the regulation of the metallothionein (Mt) promoter were generated. Mammary glands from virgin female transgenic mice at the ages of 12, 24, and 36 weeks were used to compare the effect of hPRL and G129R in various developmental stages. Mammary gland whole mount comparisons between transgenic mice and their littermates revealed a significant increase in ductal branching and lobular bud formation in hPRL transgenic mice; whereas a drastic decrease in ductal branching and lobular bud formation was observed in the mammary glands of G129R transgenic mice. In addition, total RNA isolated from the mammary glands of transgenic mice at the three different ages was analyzed on Affymetrix GeneChip Mouse Expression 430A chips (MOE430A). Microarray data revealed alteration to the gene expression levels, greatest at 12 and 36 weeks. Furthermore, hPRL and G129R transgenic mice, as well as their littermates, were treated with multiple doses of DMBA and the rate of mammary tumor formation and survival were compared. The tumor rates in the G129R transgenic mice were significantly reduced (18% at 28 weeks) as compared to that of either NTG (39%) or hPRL (40%). On the other hand, the tumor appearance is significantly earlier in the PRL transgenic group as compared to that of controls. Taken together, the data further confirmed the inhibitory effects of G129R in mammary gland development, which translates to a resistance to DMBA-initiated breast tumorigenesis.     

A new player in oncogenesis: AUF1/hnRNPD overexpression leads to tumorigenesis in transgenic mice

AUF1/heterogeneous nuclear ribonucleoprotein D (hnRNPD) binds to adenylate uridylate-rich elements contained in the 3' untranslated region of many short-lived mRNAs. This binding has been shown in vitro to control the stability of adenylate uridylate-rich element-containing mRNAs, including mRNAs encoding proto-oncogenes, cytokines, or other signaling molecules. However, no studies have yet been undertaken to identify the mRNAs subject to AUF1-mediated regulation in vivo. The purpose of our study was to investigate the biological functions of AUF1. Thus, we derived transgenic (Tg) mice, which overexpress one isoform of AUF1, the p37(AUF1). Mice of the three Tg lines analyzed exhibit altered levels of expression of several target mRNAs, such as c-myc, c-jun, c-fos, granulocyte macrophage colony-stimulating factor, and tumor necrosis factor alpha. The Tg line with the highest amount of Tg p37(AUF1) protein developed sarcomas. The tumors strongly expressed AUF1 Tg protein and Cyclin D1. Taken together, our data show that: (a) AUF1 is a key regulatory factor of gene expression in vivo; and (b) the deregulation of this heterogeneous nuclear ribonucleoprotein leads to tumorigenesis.     

Reduced lung tumorigenesis in human methylguanine DNA--methyltransferase transgenic mice achieved by expression of transgene within the target cell

Human methylguanine-DNA methyltransferase (MGMT) transgenic mice expressing high levels of O6-alkylguanine-DNA alkyltransferase (AGT) in lung were crossbred to A/J mice that are susceptible to pulmonary adenoma to study the impact of O6-methylguanine (O6mG)-DNA adduct repair on NNK-induced lung tumorigenesis. Expression of the chimeric human MGMT transgene in lung was identified by northern and western blot analysis, immunohistochemistry assay and enzymatic assay. AGT activity was 17.6 +/- 3.2 versus 1.2 +/- 0.4 fmol/microg DNA in lung of MGMT transgenic mice compared with non-transgenic mice. Immunohistochemical staining with anti-human AGT antibody showed that human AGT was expressed throughout the lung. However, some epithelial cells of bronchi and alveoli did not stain for human AGT, suggesting that the human MGMT transgene expression was heterogeneous. After 100 mg/kg NNK i.p. injection in MGMT transgenic mice, lung AGT activity remained much higher and levels of lung O6mG-DNA adducts in MGMT transgenic mice were lower than those of non-transgenic mice. In the tumorigenesis study, mice received 100 mg/kg NNK at 6 weeks of age and were killed 44 weeks later. Ten of 17 MGMT transgenic mice compared with 16 of 17 non-transgenic mice had lung tumors, P < 0.05. MGMT transgenic mice had lower multiplicity and smaller sized lung tumors than non-transgenic mice. Moreover, a reduction in the frequency of K-ras mutations in lung tumors was found in MGMT transgenic mice (6.7 versus 50% in non-transgenic mice). These results indicate that high levels of AGT expressed in mouse lung reduce lung tissue susceptibility to NNK-induced tumorigenesis due to increased repair capacity for O6mG, subsequently, decreased mutational activation of K-ras oncogene. Heterogeneity in the level of AGT expressed in different lung cell populations or other forms of carcinogenic DNA damage caused by NNK may explain the residual incidence of lung tumors in MGMT transgenic mice.     

Repeated hepatocyte injury promotes hepatic tumorigenesis in hepatitis C virus transgenic mice

Although hepatitis C virus (HCV) is a well-known causative agent of hepatocellular carcinoma (HCC), the mechanism by which HCV induces HCC remains obscure. To elucidate the role of HCV in hepatocarcinogenesis, a model of hepatocyte injury was established using HCV core transgenic mice, which were developed using C57BL/6 mice transfected with the HCV core gene under control of the serum amyloid P component promoter. After 18–24 months, neither steatosis nor hepatic tumors were found in transgenic mice. The extent of hepatocyte injury and tumorigenesis were then examined in transgenic mice following repeated administration of carbon tetrachloride (CCl₄) using various protocols (20%, 1/week; 10%, 2/week and 20%, 2/week). Serum alanine aminotransferase (ALT) levels did not differ among HCV core transgenic mice and non-transgenic littermates; however, after 40 weeks, hepatic adenomas preferentially developed in transgenic mice receiving 20% CCl₄ once weekly. Moreover, HCC was observed in transgenic mice receiving 2 weekly injections of a 20% solution of CCl₄, and was not observed in the non-transgenic control mice. In conclusion, the HCV core protein did not promote hepatic steatosis or tumor development in the absence of hepatotoxicity. However, the HCV core protein promoted adenoma and HCC development in transgenic mice following repeated CCl₄ administration. These results suggest that hepatotoxicity resulting in an increased rate of hepatocyte regeneration enhances hepatocarcinogenesis in HCV-infected livers. Furthermore, this experimental mouse model provides a valuable method with which to investigate hepatocarcinogenesis.     

Overexpression of human phosphoglycerate kinase 1 (PGK1) induces a multidrug resistance phenotype

BACKGROUND: Multidrug resistance is a significant barrier to the development of successful cancer treatment. To identify genetic alterations that are directly involved in paclitaxel resistance, a functional cloning strategy was developed. MATERIALS AND METHODS: Using mRNA from paclitaxel resistant human ovarian cancer cell line SW626TR, a cDNA library was established in a pCMV-Script vector that permits expression of cDNA inserts in mammalian cells. Transfection of the pCMV-Script/SW626TR cDNA library into the paclitaxel-sensitive human osteogenic sarcoma cell line, U-20S, resulted in several paclitaxel-resistant clones. RESULTS: DNA sequencing of clone C16 demonstrates complete homology to human phosphoglycerate kinase 1 (PGK1). Retransfection of the PGK1 insert into U-20S confers a multidrug resistant phenotype, characterized by a 30-fold increase in paclitaxel resistance, and cross-resistance to vincristine; adriamycin and mitoxantrone, but not methotrexate or cisplatin. Enzymatic analysis of the PGK1 transfectants demonstrates an increase in PGK1 activity as compared to the parental cell line, U-20S. Northern and Western analysis of PGK1 transfectants reveals no change in MDR-1 expression compared with the parental cell line. In addition, co-culture of PGK1 transfectants with verapamil only partially reverses the multidrug resistant phenotype. Rhodamine 123 studies are also consistent with an MDR-1 independent mechanism of increased drug efflux. CONCLUSION: Together this data suggests that PGK1 can induce a multidrug resistant phenotype through an MDR-1 independent mechanism.