Retinoic acid inhibits transformation by preventing phosphatidylinositol 3-kinase dependent activation of the c-fos promoter.

Retinoic acid inhibits transformation of cells by polyoma virus middle T oncoprotein. Inhibition of transformation results from a retinoic acid-dependent failure of cells to fully express the c-fos proto-oncogene. Retinoic acid prevents transactivation of the c-fos promoter by disrupting signaling between tyrosine kinases at the plasma membrane and trans-acting factors at the c-fos promoter. We used complementary genetic, biochemical and molecular approaches to demonstrate that: (1) phosphatidylinositol 3-kinase signaling is the principle mechanism of polyoma virus middle T oncoprotein activation of c-fos expression; (2) middle T/phosphatidylinositol 3-kinase transactivation of the c-fos promoter and transformation of cells requires activation of both the small GTP-binding protein Rac and Jun N-terminal kinase; (3) retinoic acid inhibits activation of Jun N-terminal kinase, thereby preventing c-fos transactivation and transformation; and (4) middle T activation of c-fos transcription requires both the serum response element and the promoter proximal cyclic AMP response element. These studies identify a novel target through which retinoids prevent oncogenic transformation.

Retinoic acid suppresses polyoma virus transformation by inhibiting transcription of the c-fos proto-oncogene.

In a previous paper, we predicted that retinoic acid suppressed polyoma virus transformation of rat F111 fibroblasts by affecting the expression of one or more genes that are involved in signalling pathways normally activated by the viral mT oncogene (Talmage & Lackey, Oncogene 7, 1837-1845, 1992). We had identified the cellular c-fos proto-oncogene as a possible candidate target for both polyoma virus mT and retinoic acid regulated expression. In this report we present the results of experiments that demonstrate that retinoic acid does indeed inhibit transcriptional transactivation of the c-fos promoter by polyoma virus, as well as by calf serum and purified serum growth factors. Further experiments demonstrate that inhibition of c-fos expression with antisense fos RNA also prevents polyoma virus induced transformation. Restoration of c-fos expression, even in the presence of retinoic acid, restored transformation, indicating that retinoic acid inhibition of c-fos expression is sufficient to explain the retinoid suppression of transformation. These results identify the c-fos proto-oncogene as a key nuclear target for mT-dependent transformation and show that the anticarcinogenic properties of retinoic acid can be brought about by inhibiting c-fos expression.

Uptake of antisense oligonucleotides and functional block of acetylcholine receptor subunit gene expression in primary embryonic neurons.

Several recent studies have used antisense oligonucleotides in the nervous system to probe the functional role of particular gene products. Since antisense oligonucleotide-mediated block of gene expression typically involves uptake of the oligonucleotides, we have characterized the mechanism of this uptake into developing neurons from embryonic chickens. Antisense oligonucleotides (15 mers) added to the bathing media are taken up into the embryonic chicken sympathetic neurons maintained in vitro. A portion of the oligonucleotide uptake is temperature dependent and saturates at extracellular oligonucleotide concentrations > or = 20 microM. This temperature sensitive, saturable component is effectively completed by single nucleotides of ATP and AMP and is reminiscent of receptor-mediated endocytosis of oligonucleotides described in non-neuronal cells. The efficiency of the oligonucleotide uptake system is dependent on the developmental stage of the animal but independent of the number of days that the neurons are maintained in vitro. Following the uptake of antisense oligonucleotides directed against ion channel subunit genes expressed by these neurons (nicotinic acetylcholine receptor subunit alpha 3; nAChR alpha 3), biophysical assays reveal that the functional expression of the target gene is largely blocked. Thus the number of wild type nAChR channels expressed is decreased by approximately 80%-90%. Furthermore, following antisense deletion of alpha 3, "mutant" nAChRs with distinct functional characteristics are expressed. In sum, these studies characterize the uptake of antisense oligonucleotide and demonstrate the functional block of specific gene expression in primary developing neurons.(ABSTRACT TRUNCATED AT 250 WORDS)

Functional contribution of neuronal AChR subunits revealed by antisense oligonucleotides.

Although multiple related genes encoding nicotinic acetylcholine receptor (AChR) subunits have been identified, how each of these subunits contributes to AChRs in neurons is not known. Sympathetic neurons express four classes of AChR channels and six AChR subunit genes (alpha 3, alpha 4, alpha 5, alpha 7, beta 2, and beta 4). The contribution of individual subunits to AChR channel subtypes in these neurons was examined by selective deletion with antisense oligonucleotides. An alpha 3 antisense oligonucleotide decreased the number and altered the properties of the normally expressed ACh-activated channels. The remaining AChR channels have distinct biophysical and pharmacological properties that indicate an important functional contribution of the alpha 7 subunit.

Developmental changes in transmitter sensitivity and synaptic transmission in embryonic chicken sympathetic neurons innervated in vitro.

Dispersed neurons from embryonic chicken sympathetic ganglia were innervated in vitro by explants of spinal cord containing the autonomic preganglionic nucleus or somatic motor nucleus. The maturation of postsynaptic acetylcholine (ACh) sensitivity and synaptic activity was evaluated from ACh and synaptically evoked currents in voltage-clamped neurons at several stages of innervation. All innervated cells are more sensitive to ACh than uninnervated neurons regardless of the source of cholinergic input. Similarly, medium conditioned by either dorsal or ventral explants mimics innervation by enhancing neuronal ACh sensitivity. This increase is due to changes in the rate of appearance of ACh receptors on the cell surface. There are also several changes in the nature of synaptic transmission with development in vitro, including an increased frequency of synaptic events and the appearance of larger amplitude synaptic currents. In addition, the mean amplitude of the unit synaptic current mode increases, as predicted from the observed changes in postsynaptic sensitivity. Although spontaneous synaptic current amplitude histograms with multimodal distributions are seen at all stages of development, histograms from early synapses are typically unimodal. Changes in the synaptic currents and ACh sensitivity between 1 and 4 days of innervation were paralleled by an increase in the number of synaptic events that evoked suprathreshold activity in the postsynaptic neurons. The early pre- and postsynaptic differentiation described here for interneuronal synapses formed in vitro may be responsible for increased efficacy of synaptic transmission during development in vivo.

Modulation of hnRNP A1 protein gene expression by epidermal growth factor in Rat-1 cells.

The levels of mRNA encoding hnRNP core protein A1 have been compared in quiescent and proliferating Rat-1 embryonic fibroblasts. Northern blot hybridization analyses using probes made from an A1 cDNA clone, lambda HDP-182, isolated by Cobianchi et al. (J. Biol. Chem. 261:3536-2543 (1986] indicated that three sizes of poly A + RNAs, 1.6, 2.0, & 4.0 kb, have extensive sequence homology. The levels of all three A1 RNA species are responsive to the proliferation state of the cells. Stimulation of quiescent Rat-1 cells with serum or epidermal growth factor resulted in a 2- to 5-fold increase in the levels of each of these three RNAs that was evident after 2 hours and reached a peak after about 8 hours. Addition of the protein synthesis inhibitor, cycloheximide, along with epidermal growth factor completely blocked the upsurge in A1 RNA levels. Thus, the A1 RNA species are not primary transcriptional targets of epidermal growth factor but do show an induction pattern similar to mRNAs encoding some glycolytic enzymes.