Genetic analysis in Caenorhabditis elegans has uncovered essential roles for DAF-16 in longevity, metabolism, and reproduction. The mammalian orthologs of DAF-16, the closely-related FOXO subclass of forkhead transcription factors (FKHR͞FOXO1, FKHRL1͞FOXO3a, and AFX͞FOXO4), also have important roles in cell cycle arrest, apoptosis and stress responses in vitro, but their in vivo physiological roles are largely unknown. To elucidate their role in normal development and physiology, we disrupted each of the Foxo genes in mice. Foxo1-null embryos died on embryonic day 10.5 as a consequence of incomplete vascular development. Foxo1-null embryonic and yolk sac vessels were not well developed at embryonic day 9.5, and Foxo1 expression was found in a variety of embryonic vessels, suggesting a crucial role of this transcription factor in vascular formation. On the other hand, both Foxo3a-and Foxo4-null mice were viable and grossly indistinguishable from their littermate controls, indicating dispensability of these two members of the Foxo transcription factor family for normal vascular development. Foxo3a-null females showed age-dependent infertility and had abnormal ovarian follicular development. In contrast, histological analyses of Foxo4-null mice did not identify any consistent abnormalities. These results demonstrate that the physiological roles of Foxo genes are functionally diverse in mammals.
Mesenchymal stem cells (MSCs) are bone marrow stroma-derived cells, which can differentiate into several types of mesenchymal tissues. Although regarded as tissue-specific stem cells, human MSCs (hMSCs) have a low proliferative ability with a finite life span, which is a hurdle to further analysis of their biology. Here we attempted to establish immortalized hMSCs by retrovirus-mediated gene transfer. The gain in telomerase activity obtained on expression of human telomerase reverse transcriptase (hTERT) was found not to be enough to make the cell line immortal. A combination of hTERT with human papillomavirus E6 and E7 successfully immortalized hMSCs without affecting the potential for adipogenic, osteogenic, and chondrogenic differentiation. From the parental immortalized hMSC, 100 single-cell derived clones were established, of which the differentiation properties varied considerably, including tri-, bi-, and uni-directional clones, suggesting that hMSCs are constituted by a group of cells with different differentiation potential. These cell lines, being the first established immortalized clonal cell lines of hMSCs, could provide insights into the mechanisms regulating the early steps of differentiation from undifferentiated MSCs into a specific lineage.
We report here that the Id2 (inhibitor of DNA binding 2) gene is a novel target of transcriptional activation by EWS -FLI1 and EWS -ERG, two fusion proteins that characterize Ewing family tumors (EFTs). To identify downstream targets of these EWS -ETS fusion proteins, we introduced EWS -ETS fusion constructs into a human fibrosarcoma cell line by retroviral transduction. cDNA microarray analysis revealed that Id2 expression was up-regulated by introducing the EWS -ETS fusion gene but not by the normal full-length ETS gene. An Id2 promoter-luciferase reporter assay showed that transactivation by EWS -ETS involves the minimal Id2 promoter and may function in cooperation with c-Myc within the full-length regulatory region. A chromatin immunoprecipitation assay revealed direct interaction between the Id2 promoter and EWS-FLI1 fusion protein in vivo. Significantly higher expression of Id2 and c-Myc was observed in all of the six EFT cell lines examined compared to six other sarcoma cell lines. Moreover, high levels of Id2 expression were also observed in five of the six primary tumors examined. Id2 is generally thought to affect the balance between cell differentiation and proliferation in development and is highly expressed in several cancer types. Considering these previous studies, our data suggest that the oncogenic effect of EWS -ETS may be mediated in part by up-regulating Id2 expression.
Fusion of TLS/FUS and CHOP gene by reciprocal translocation t(12;16)(q32;q16) is a common genetic event found in myxoid and round-cell liposarcomas. Characterization of this genetic event was performed by three methods, Southern blot, RT ± PCR, and genomic long-distance PCR in nine myxoid and three round-cell liposarcomas. All but one tumors showed genetic alternations indicating the fusion of TLS/FUS and CHOP gene. Two novel types of fusion transcripts were found, of which one lacked exon 2 sequence of CHOP gene, and the other lacked 3' half of exon 5 of TLS gene. The latter case was caused by a cryptic splicing site which was created by the genomic fusion. Detailed analyses genomic fusion points revealed several sequence characteristics surrounding the fusion points. Homology analyses of breakpoint sequences with known sequence motifs possibly involve in the process of translocation uncovered Translin binding sequences at both of TLS/ FUS and CHOP breakpoints in two cases. Translocations were always associated with other genetic alterations, such as deletions, duplications, or insertions. Short direct repeats were almost always found at both ends of deleted or duplicated fragments some of which had apparently been created by joining of sequences that ank the rearrangement. Finally, consensus topoisomerase II cleavage sites were found at breakpoints in all cases analysed, suggesting a role of this enzyme in creating staggered ends at the breakpoint. These data suggested that sequence characteristics may play an important role to recruit several factors such as Translin and topoisomerase II in the process of chromosomal translation in liposarcomas.
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