Murine FGF-12 and FGF-13: expression in embryonic nervous system, connective tissue and heart.

The molecular cloning of cDNAs encoding murine fibroblast growth factor-13 (FGF-13/FHF-2) and three isoforms of murine FGF-12 (FHF-1) is described. Like their highly conserved human counterparts, murine FGF-12 and FGF-13 are part of a distinct subfamily of FGF-like proteins characterized by a greater degree of amino acid sequence cross-homology and by conserved N-terminal domains which do not include secretion signal sequences. In addition to their expression in several adult tissues, both of these FGF genes are prominently and regionally expressed in midgestation mouse embryos, as revealed by in situ hybridization. Fgf12 and fgf13. RNAs were detected in developing central nervous system in cells outside the proliferating ependymal layer, and fgf13 RNA was also found throughout the peripheral nervous system. Fgf12 is expressed in developing soft connective tissue of the limb skeleton and in presumptive connective tissue linking vertebrae and ribs. Both FGF genes are also expressed in the myocardium of the heart, with fgf12 RNA found only in the atrial chamber and fgf13 RNA detected in both atrium and ventricle. On the basis of their novel structure and patterns of expression, FGF-12 and FGF-13 are anticipated to perform embryonic functions distinct from other known FGF molecules.

Chromosomal mapping of two novel human FGF genes, FGF11 and FGF12.

The fibroblast growth factor (FGF) family comprises to date 12 members, which are involved in various physiological processes throughout embryogenesis and adult life. Two novel members of the family have been identified recently (FGF11 and FGF12). Using in situ hybridization on metaphasic chromosomes, we have been able to assign FGF11 to band p12-p13 of human chromosome 17 and FGF12 to band q28 of human chromosome 3.

Establishment of permanent wild-mouse cell lines with readily identifiable marker chromosomes.

Physical gene mapping by in situ hybridization is a difficult task in an all-acrocentric mouse karyotype, because all of the chromosomes are morphologically very similar. These difficulties can be overcome by using the many different metacentric Robertsonian translocation (Rb) chromosomes derived from wild mice. Here we describe the establishment of two Moloney murine leukemia virus-transformed suspension cell lines, WMP-1 and WMP-2, derived from wild mice of the strain WMP/WMP. These mice carry nine pairs of metacentric Rb chromosomes containing chromosomes 1 to 18. Chromosome 19 and the sex chromosomes are the only acrocentric chromosomes. Furthermore, a heterozygous reciprocal translocation between chromosomes 13 and 17 involved in two Rb chromosomes is present in this stock and provides additional marker chromosomes. The chromosome designation of these mice is Rb(10.17)9Mpl Rb(13.15)10Mpl T(13.17)1Lub.

Cyclin D1/bcl-1 cooperates with myc genes in the generation of B-cell lymphoma in transgenic mice.

The chromosomal translocation t(11:14) is associated with human lymphoid neoplasia affecting centrocytic B-cells of intermediate differentiation. As a consequence the cyclin D1 (bcl-1) gene is juxtaposed to the immunoglobulin heavy chain enhancer E mu. To show that transcriptional activation of cyclin D1 is causally involved in the generation of B-cell neoplasia we have generated transgenic mice that carry a cyclin D1 gene under the transcriptional control of the E mu element. E mu cyclin D1 transgenic mice show only very subtle alterations in the cycling behaviour of B-cell populations in the bone marrow compared with normal mice and do not develop lymphoid tumours. However, E mu-directed coexpression of cyclin D1 and N-MYC or L-MYC in double transgenic mice reveals a strong cooperative effect between MYC and cyclin D1 provoking the rapid development of clonal pre-B and B-cell lymphomas. Interestingly, crossing of cyclin D1 transgenic mice with E mu L-myc transgenics that express their transgene in both B- and T-cells but predominantly develop T-cell tumours leads in double transgenics exclusively to B-cell neoplasia. The data presented here demonstrate that transcriptional activation of cyclin D1 can oncogenically transform B-cells in concert with a myc gene. They establish cyclin D1 as a proto-oncogene whose activity appears to depend on a specific cell type as well as on a specific cooperating partner and link disturbances in the regulation of cell cycle progression to the development of human malignancies.

E2F-dependent regulation of human MYC: trans-activation by cyclins D1 and A overrides tumour suppressor protein functions.

Transcription of the human proto-oncogene MYC is repressed in quiescent or non-dividing cells. Upon mitogenic stimulation expression of MYC is rapidly and transiently induced, maintained throughout G1, and declines to a basal level throughout further cell cycle transitions. Regulation of MYC promoter activity critically depends on the presence of a binding site for transcription factor E2F. Human E2F has been implicated also in the control of cell cycle progression through it association with the retinoblastoma suppressor gene product RB, and with multimeric protein complexes containing the G1-S- and S-phase cyclins E and A, respectively. Using CAT reporter co-transfection assays we show here that transcription from the MYC P2 promoter is induced efficiently by E2F-1, but repressed by RB. Furthermore, overexpression of cyclin A strongly activates the MYC promoter and this effect is further enhanced by coexpression of E2F-1 and cyclin A. We also find that expression of G1-phase cyclin D1 leads to an E2F binding site-dependent trans-activation of the MYC promoter and that this activation can be abrogated by overexpression of RB. The interaction of D-type G1 cyclins with RB resembles that of the adenovirus E1A protein with RB in that it can disrupt inhibitory E2F-RB complexes. Our results support a model in which intervention of distinct cyclins and their respective associated kinases promotes transcriptional activation of MYC throughout the cell cycle either by conversion of E2F within multimeric complexes into an active transcription factor or by liberation of free functional E2F.

Oncogenic activity of cyclin D1 revealed through cooperation with Ha-ras: link between cell cycle control and malignant transformation.

Circumstantial evidence implicates the putative cell cycle regulator cyclin D1 in the process of malignant transformation. Overexpression of cyclin D1 is observed in mammary carcinomas as a result of gene amplification and in parathyroid adenomas and centrocytic B-cell lymphomas as a consequence of chromosomal rearrangements and juxtaposition of the cyclin D1 gene to strong transcriptional control elements. These findings suggest that deregulation of cyclin D1 expression may contribute to malignant transformation in these tumours. To date, however, an oncogenic potential of cyclin D1 has not been demonstrated and the mechanism of its oncogenic activation remains obscure although overexpression of the wild-type protein is likely. We report here that the overexpression of cyclin D1 induces transformation in primary rat embryo fibroblasts in cooperation with activated Ha-ras. Cyclin D1/Ha-ras transformed cells are immortalized, show anchorage independence and give rise to fibrosarcomas in nude mice. Our data directly demonstrate that cyclin D1 is a proto-oncogene that can be activated by transcriptional deregulation. Its previously demonstrated ability to interact with putative cell cycle regulators suggests that cyclin D1 defines a new class of proto-oncogenes.