Search

We used fibrosarcoma from an H2-K/v-juntransgenic mouse to derive a series of three immortal cell lines (JUN-1, -2, and -3). The cell lines exhibit strikingly different behavior regarding phenotype transformation. Features examined include contact inhibition and density limitation of growth, proliferation, invasiveness, motility, and organization of the microfilament system. Overall, JUN-2 and JUN-3 represent extreme phenotypes, with JUN-2 having a phenotype indicative of low-level cellular transformation and JUN-3 meeting all the criteria of the transformed phenotype. JUN-1 cells can also be regarded as transformed, but to a lesser extent than JUN-3. Their phenotype is in the majority of characteristics intermediary between JUN-2 and JUN-3. The transformation status is inversely related to the expression of the v-juntransgene, which is the highest in JUN-2, lower in JUN-1 and very low in JUN-3. This might be related to the MHC class I promoter driving its expression and to the general observation of repression of MHC class I genes coupled with cellular transformation. Based on this premise, we present a model of H2-K/v-jun-mediated tumorigenesis, in which v-jun-conditioned transformation represents merely an initial phase of tumorigenesis. Later during tumor progression, additional oncogenes are activated and/or tumor suppressor genes inactivated, leading on the one hand to further exacerbation of the transformed phenotype, and on the other hand to the repression of the H2-K/v-juntransgene (fixed in JUN-3). We believe that the system of JUN cell lines can be valuable for further molecular analysis of transformation-related traits.

There is evidence that the profilin:actin complex is the immediate precursor in the formation of actin filaments in cells. This paper describes the cell morphology and microfilament distribution after microinjection of covalently cross-linked profilin:β/γ-actin (PxA) in two different cell lines. Injected cells were either kept unstimulated or stimulated with platelet-derived growth factor (PDGF) before fixation and visualization of filamentous actin. After injection of low doses of PxA, the cells displayed an actin organization characterized by a clearance of diffuse fluorescence from a region immediately interior of ruffling edges and the appearance of small dots of fluorescence in the same region. At higher concentrations, PxA effectively inhibited outgrowth of lamellae and microspikes, and there was a drastic reduction of actin staining in the zone behind the advancing edge. This effect is reminiscent of the effect of cytochalasin B on fibroblasts and the growth cone of neuronal cells. As in these cases, there remained a rim of actin-dependent fluorescence on the very edge of the membrane lamella, particularly in the PxA-treated fibroblasts. The interference of PxA with the formation of surface structures was pronounced after PDGF stimulation. Here, PxA effectively eliminated the enhancement of the ruffling activity in the cell edges and on the dorsal surface of the cells. In contrast to PxA, injection of non-cross-linked profilin:β/γ-actin had no apparent effect on cell morphology and microfilament distribution except for an increased concentration of filamentous actin in one of the cell lines.

Phosphoinositide 3´-kinases constitute a family of lipid kinases implicated in signal transduction through tyrosine kinase receptors and heterotrimeric G protein-linked receptors. Phosphoinositide 3´-kinases that bind to the platelet-derived growth factor receptor are composed of two subunits: the p85 subunit acts as an adapter and couples the catalytic p110 subunit to the activated receptor. There are different isoforms of p85 as well as of p110, the individual roles of which have been elusive. Using microinjection of inhibitory antibodies specific for either p110α or p110β we have investigated the involvement of the two p110 isoforms in platelet-derived growth factor- and insulin-induced actin reorganization in porcine aortic endothelial cells. We have found that antibodies against p110α, but not antibodies against p110β, inhibit platelet-derived growth factor-stimulated actin reorganization, whereas the reverse is true for inhibition of insulin-induced actin reorganization. These data indicate that the two phosphoinositide 3´-kinase isoforms have distinct roles in signal transduction pathways induced by platelet-derived growth factor and insulin.

We are investigating structure–function relationships in profilin and actin by site-specific mutagenesis using a yeast,Saccharomyces cerevisiae,expression system to produce wild-type and mutant proteins. This paper shows that deleting proline 96 and threonine 97, which are located close to the major actin binding site on profilin, did not significantly alter the interaction between profilin and phosphatidylinositol 4,5-bisphosphate, nor did it affect the profilin:poly(l-proline) interaction. The mutant protein, however, had a lower capacity to bind to actinin vitrothan wild-type profilin, though it showed a slightly increased profilin-enhanced nucleotide exchange on the actin. When microinjected into Swiss 3T3 mouse fibroblasts or porcine aortic endothelial cells, the mutant profilin did not change the organization of the microfilament system like the wild-type profilin did. This provides further evidence that profilin controls microfilament organization in the cell by interacting directly with actin.