No abstract

A family of structurally related proteins homologous to the Drosophila mothers against dpp (MAD) gene product have been implicated in signal transduction by members of the TGF-b superfamily. One of these MAD related proteins (DPC4) has been cloned as a candidate tumour suppressor in pancreas carcinomas, suggesting a role for DPC4 in growth regulation by TGF-b related proteins. The involvement of DPC4 in TGF-b1 induced growth inhibition and transcriptional response is demonstrated here, by the introduction of DPC4 in the TGF-b and activin insensitive breast tumour cell line MDA-MB-468, from which the DPC4 gene is deleted. Transfection of DPC4 in this cell line restores both growth inhibition and the induction of a TGF-b sensitive reporter construct (3TPlux) by TGF-b1. In contrast, a DPC4 splice variant lacking amino acid residues 223 ± 301 and cloned from another TGF-b and activin resistant breast tumour cell line (MDA-MB-231), does not restore the induction of the 3TPlux reporter by TGF-b1. We also show that in this latter cell line activin resistance is partly due to the absence of a functional activin type IB receptor. These results indicate that DPC4 is part of the TGF-b signalling cascade and mediates TGF-b induced growth inhibition. Together with the deletion of DPC4 from pancreas carcinomas these results suggest a role for DPC4 as a tumour suppressor.

No abstract

In a search for functions of transforming growth factor-beta during early embryonic development we used two different experimental approaches. In the first we made use of embryonic stem (ES) cells. ES cells in culture differentiate to derivatives of all three germ layers and mimic some aspects of organogenesis when grown as aggregates in suspension to form embryoid bodies. Differentiation proceeds further when the embryoid bodies attach to suitable substrates. Muscle and neuronal cells are among the most readily identified cell types then formed. We examined the effect of all-trans retinoic acid (RA) and members of the transforming growth factor-beta family (TGF-beta 1, TGF-beta 2) under these conditions in an assay where single aggregates formed in hanging microdrops in medium supplemented with serum depleted of lipophilic substances which would include retinoids. Endoderm-like cells formed under all conditions tested. RA at concentrations of 10(-8) M and 10(-7) M induced the formation of neurons but in the absence of RA or at concentrations up to 10(-9) M, neurons were not observed. Instead, beating muscle formed in about one-third of the plated aggregates; this was greatly reduced when RA concentrations increased above 10(-9) M. Immunofluorescent staining for muscle specific myosin showed that two muscle cell types could be distinguished: elongated, non-contractile myoblasts and mononucleate flat cells. The mononucleate flat cells appeared to correspond with rhythmically contracting muscle. The number of non-contractile myoblasts increased 3-fold over controls in the presence of 10(-9) M RA. TGF-beta s increased the number of contractile and non-contractile muscle cells by a factor 3 to 7 over controls, depending on the TGF-beta isoform added and the muscle cell type formed. TGF-beta 2 also invariably increased the rate at which contracting muscle cells were first observed in replated aggregates. The stimulatory effect of TGF-beta s on the formation of mononucleate flat cells was completely abrogated by RA at 10(-9) M while the number of myoblasts under similar conditions was unchanged. These data suggest that a complex interplay between retinoids and TGF-beta isoforms may be involved in regulation of differentiation in early myogenesis. In the second approach, neutralizing polyclonal rabbit antibodies specific for TGF-beta 2 were injected into the cavity of mouse blastocysts 3.5 days post coîtum (pc). After 1 day in culture, embryos were transferred to pseudopregnant females. The number of decidua, embryos and resorptions were counted at day 8.5-9.5 pc.(ABSTRACT TRUNCATED AT 400 WORDS)