Brain cells from murine embryos were transfected with the polyoma virus large T or the adenovirus 5 EIA gene and, simultaneously, with the phosphotransferase coding NeoR gene. The efficiently transfected cells were selected for their resistance to Geneticin (G418) and their ability to clone at low cell density. Subsequently, most of the selected cells could be sub‐cloned and continuously grown for 6‐18 months so far. Their doubling time varied between 18 and 72 h. From independent transfections, more than one hundred cell lines were established. They did not exhibit a transformed phenotype, but subsequent transfection with the polyoma middle T gene induced their oncogenic transformation. The maintenance and expression of the transferred genes were verified. Most of the analyzed cell lines retained glial properties. These results suggest that the lines obtained as well as a further extension of this in vitro system should be of interest for the study of nervous cell interactions, differentiation and functions.
Immortalized neural precursor cell lines carrying the polyoma large tumor (T) gene have been shown previously to retain a clear-cut contact inhibition of growth and to differentiate in vitro. In the present study, we have identified and isolated cDNA clones corresponding to RNA expressed preferentially when these cells reach confluence. One of them, NPDC-1, is expressed specifically in the nervous system. The transfection of dividing cells with a NPDC-1 expression vector results in the inhibition of cell proliferation. In addition, the stable introduction of NPDC-1 into transformed cells, even of nonneural origin, leads to the suppression of transformed characteristics.The identification of transcriptional factors has improved our knowledge of mechanisms underlying mammalian development and cell-lineage determination (for reviews, see refs.
We have previously identi®ed NPDC-1, a neural factor involved in the control of proliferation and dierentiation, and we have shown that the stable introduction of NPDC-1 into transformed cells down-regulates cell proliferation both by increasing the generation time and by suppressing transformed properties. The data presented here indicate that, in vitro, NPDC-1 is able to interact with the transcription factor E2F-1 and some cell cycle proteins, such as D-cyclins and cdk2. In addition, two-hybrid experiments in mammalian cells show that the interaction between NPDC-1 and E2F-1 can also occur in vivo. This interaction reduces the binding of E2F-1 to DNA and its transcriptional activity. Taken together, the data suggest that NPDC-1 could in¯uence cell cycle progression and neural dierentiation through its association with E2F-1. Oncogene (2000) 19, 5000 ± 5009.
We have investigated the expression of macrophage-colony stimulating factor (M-CSF) gene in mouse brain during development. Northern blot analysis of cerebral RNA evidenced a 4.5-kb M-CSF transcript from day 14 of gestation until 2 weeks after birth. The cell type responsible for this transcription was studied using in vitro cell cultures. The 4.5-kb M-CSF transcript was found both in astrocyte primary cultures and in immortalized astrocytic cell lines. M-CSF mRNA was also detected in lipopolysaccharide-stimulated brain macrophage cultures. These results suggest that M-CSF is involved in the outgrowth of microglia during ontogenesis.
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