Chronic myeloid leukemia with increased granulocyte progenitors in mice lacking junB expression in the myeloid lineage.

The functions of JunB during myelopoiesis were studied in vivo. Transgenic mice specifically lacking JunB expression in the myeloid lineage (junB(-/-)Ubi-junB mice) develop a transplantable myeloproliferative disease eventually progressing to blast crisis, which resembles human chronic myeloid leukemia. Similarly, mice reconstituted with ES cell-derived junB-/- fetal liver cells also develop a myeloproliferative disease. In both cases, the absence of JunB expression results in increased numbers of granulocyte progenitors, which display enhanced GM-CSF-mediated proliferation and extended survival, associated with changes in the expression levels of the GM-CSFalpha receptor, the anti-apoptotic proteins Bcl2 and Bclx, and the cell cycle regulators p16(INK4a) and c-Jun. Importantly, ectopic expression of JunB fully reverts the immature and hyperproliferative phenotype of JunB-deficient myeloid cells. These results identify JunB as a key transcriptional regulator of myelopoiesis and a potential tumor suppressor gene.

Formation of transformed endothelial cells in the absence of VEGFR-2/Flk-1 by Polyoma middle T oncogene.

The middle T antigen of murine Polyomavirus (PymT) rapidly transforms endothelial cells leading to vascular malformations reminiscent of endothelial tumors or hemangiomas. Flk-1, a receptor tyrosine kinase which is activated upon binding of its ligand VEGF, is predominantly expressed in endothelial cells and essential for the formation of blood vessels since absence of Flk-1 prevents the development of mature endothelial cells in mice and in ES-cell differentiation experiments. To investigate the role of Flk-1 in PymT-induced vascular tumor formation, we studied the expression of Flk-1 and VEGF in PymT-transformed endothelial cells (Endothelioma cells, END. cells). The receptor and its ligand were both expressed in END. cells suggesting that a VEGF/Flk-1 autocrine loop might be causally involved in the formation of vascular tumors. To test this hypothesis, ES cells lacking Flk-1 were generated and the transforming potential of PymT was analysed after in vitro differentiation. Flk-1(-/-) END. cell lines were established which are morphologically identical to flk-1(+/+) END. cells and which express several markers characteristic for endothelial cells. This result suggests that PymT functionally replaces the requirement of Flk-1 in expansion and/or survival of endothelial progenitor cells. Therefore, flk-1(-/-) END. cells provide a powerful tool to dissect the downstream signaling pathways of Flk-1.

Control of cell cycle progression by c-Jun is p53 dependent.

The c-jun proto-oncogene encodes a component of the mitogen-inducible immediate-early transcription factor AP-1 and has been implicated as a positive regulator of cell proliferation and G1-to-S-phase progression. Here we report that fibroblasts derived from c-jun-/- mouse fetuses exhibit a severe proliferation defect and undergo a prolonged crisis before spontaneous immortalization. The cyclin D1- and cyclin E-dependent kinases (CDKs) and transcription factor E2F are poorly activated, resulting in inefficient G1-to-S-phase progression. Furthermore, the absence of c-Jun results in elevated expression of the tumor suppressor gene p53 and its target gene, the CDK inhibitor p21, whereas overexpression of c-Jun represses p53 and p21 expression and accelerates cell proliferation. Surprisingly, protein stabilization, the common mechanism of p53 regulation, is not involved in up-regulation of p53 in c-jun-/- fibroblasts. Rather, c-Jun regulates transcription of p53 negatively by direct binding to a variant AP-1 site in the p53 promoter. Importantly, deletion of p53 abrogates all defects of cells lacking c-Jun in cell cycle progression, proliferation, immortalization, and activation of G1 CDKs and E2F. These results demonstrate that an essential, rate-limiting function of c-Jun in fibroblast proliferation is negative regulation of p53 expression, and establish a mechanistic link between c-Jun-dependent mitogenic signaling and cell-cycle regulation.

Polyoma middle T-induced vascular tumor formation: the role of the plasminogen activator/plasmin system.

The middle T antigen of murine Polyomavirus (PymT) rapidly transforms endothelial cells, leading to the formation of vascular tumors in newborn mice. Transformed endothelial (End.) cell lines established from such tumors exhibit altered proteolytic activity as a result of increased expression of urokinase-type plasminogen activator (uPA) and are capable of inducing vascular tumors efficiently when injected into adult mice. In this study we have used mice lacking components of the PA/plasmin system to analyze the role of this system in the transformation process and in tumor growth. We found that the proteolytic status of the host is not a critical determinant for PymT-induced vascular tumor formation. In addition, the lack of either uPA or tissue-type PA (tPA) activity is not limiting for the establishment and proliferation of End. cells in vitro, although the combined loss of both PA activities leads to a marked reduction in proliferation rates. Furthermore, the in vitro morphogenetic properties of mutant End. cells in fibrin gels could only be correlated with an altered proteolytic status in cells lacking both uPA and tPA. However, in contrast with tumors induced by PymT itself, the tumorigenic potential of mutant and wild-type End. cell lines was found to be highly dependent on the proteolytic status of both the tumor cells and the host. Thus, genetic alterations in the PA/plasmin system affect vascular tumor development, indicating that this system is a causal component in PymTmediated oncogenesis.

Cell proliferation and cell cycle progression are not impaired in fibroblasts and ES cells lacking c-Fos.

The transcription factor AP-1 is thought to play an important role in the control of cell proliferation, but the function of individual Fos and Jun family members is a largely unresolved issue. To directly analyse the function of c-Fos in the control of cell proliferation we have used embryonic stem (ES) cells and fibroblasts lacking c-Fos due to a disruption of the c-fos gene by homologous recombination. Our results demonstrate that proliferation of normally cycling cells and reentry of quiescent cells into the cell cycle following serum stimulation are not c-Fos-dependent and occur with similar efficiency in c-fos-/- and control cells. We also show that there is no compensatory overexpression or activation of other known Fos or Jun family members. On the contrary, the c-fos-/- cells showed a reduced induction of fra-1 after serum stimulation which is in agreement with the previous identification of fra-1 as a c-Fos target gene. Comparison of the AP-1 binding and transactivation activities in c-fos-/- and +/+ fibroblasts by electrophoretic mobility antibody supershift and CAT assays suggests that c-Fos is not a major component of AP-1 complexes in these cells. It is therefore conceivable that the lack of any detectable effect on cell proliferation in c-fos-/- cells might be due to a functional redundancy among the different AP-1 family members.

Bone and haematopoietic defects in mice lacking c-fos.

The proto-oncogene c-fos is the cellular homologue of v-fos originally isolated from murine osteosarcoma. Fos protein is a major component of the AP-1 transcription factor complex, which includes members of the jun family. Stable expression of c-fos in mice has been demonstrated in developing bones and teeth, haematopoietic cells, germ cells and in the central nervous system. It has been proposed that c-fos has an important role in signal transduction, cell proliferation and differentiation. We have previously demonstrated that overexpression of c-fos in transgenic and chimaeric mice specifically affects bone, cartilage and haematopoietic cell development. To understand better the function of c-fos in vivo, we used gene targeting in embryonic stem cells to generate cells and mice lacking c-fos. Here we report that heterozygous fos +/- mice appear normal, although females exhibit a distorted transmission frequency. All homozygous fos -/- mice are growth-retarded, develop osteopetrosis with deficiencies in bone remodelling and tooth eruption, and have altered haematopoiesis. These data define the c-Fos protein as an essential molecule for the development of specific cellular compartments.

A novel target cell for c-fos-induced oncogenesis: development of chondrogenic tumours in embryonic stem cell chimeras.

Embryonic stem (ES) cells were used to investigate the target cell specificity and consequences of c-fos when expressed ectopically during embryonic development. Chimeric mice generated with different ES cell clones selected for high exogenous c-fos expression were not affected during embryonic development; however, a high frequency of cartilage tumours developed as early as 3-4 weeks of age apparently independent of the extent of chimerism. The tumours originated from cartilagenous tissues and contained many chondrocytes. Expression of exogenous c-fos RNA and Fos protein was observed during development but was highest in tumour tissues, predominantly in differentiating chondrocytes. A number of primary and clonal tumour-derived cell lines were established which expressed high levels of c-fos, c-jun as well as the cartilage-specific gene type II collagen and which gave rise to cartilage tumours in vivo, some of which also contained bone. Interestingly, the levels of c-Fos and c-Jun appeared to be coordinately regulated in the cell lines as well as in chimeric tissues. Thus, we demonstrate that chondrogenic cells and earlier progenitors are specially transformed by Fos/Jun and therefore represent a novel mesenchymal target cell for c-fos overexpression.

Increased proteolytic activity is responsible for the aberrant morphogenetic behavior of endothelial cells expressing the middle T oncogene.

Expression of the polyoma virus middle T (mT) oncogene in vivo is associated with a profound subversion of normal vascular development, which results in the formation of endothelial tumors (hemangiomas). In an attempt to understand the molecular mechanisms responsible for this phenomenon, we have investigated, in an in vitro system, the morphogenetic properties of endothelial cells expressing this oncogene. mT-expressing endothelioma (End) cells grown within fibrin gels formed large hemangioma-like cystic structures. All End cell lines examined expressed high levels of fibrinolytic activity resulting from increased production of urokinase-type plasminogen activator and decreased production of plasminogen activator inhibitors. Neutralization of excess proteolytic activity by exogenously added serine protease inhibitors corrected the aberrant in vitro behavior of End cells and allowed the formation of capillary-like tubules. These results suggest that tightly controlled proteolytic activity is essential for vascular morphogenesis and that physiological protease inhibitors play an important regulatory role in angiogenesis.