The product of the trk proto-oncogene encodes a receptor for nerve growth factor (NGF). Here we show that NGF is a powerful mitogen that can induce resting NIH 3T3 cells to enter S phase, grow in semisolid medium, and become morphologically transformed. These mitogenic effects are absolutely dependent on expression of gp140trk receptors, but do not require the presence of the previously described low affinity NGF receptor. gp140trk also serves as a receptor for the related factor neurotrophin-3 (NT-3), but not for brain-derived neurotrophic factor. Both NGF and NT-3 induce the rapid phosphorylation of gp140trk receptors and the transient expression of c-Fos proteins. However, NT-3 appears to elicit more limited mitogenic responses than NGF. These results indicate that the product of the trk proto-oncogene is sufficient to mediate signal transduction processes induced by NGF and NT-3, at least in proliferating cells.

The expression of different members of the Jun and Fos families of transcription factors is rapidly induced following serum stimulation of quiescent fibroblasts. To determine whether these proteins are required for cell cycle progression, we microinjected affinity-purified antibodies directed against c-Fos, FosB, Fra-l, c-Jun, JunB, and JunD, and antibodies that recognize either the Fos or the Jun family of proteins, into Swiss 3T3 cells and determined their effects in cell cycle progression by monitoring DNA synthesis. We found that microinjection of anti-Fos and anti-Jun family antibodies efficiently blocked the entrance to the S phase of serum-stimulated or asynchronously growing cells. However, the antibodies against single members of the Fos family only partially inhibited DNA synthesis. In contrast, all three Jun antibodies prevented DNA synthesis more effectively than did any of the anti-Fos antibodies.

We have characterized the expression of c-Jun, JunB, JunD, c-Fos, and FosB proteins following serum stimulation of quiescent Swiss 3T3 cells by immunoprecipitation analyses. The synthesis of the three Jun proteins rapidly increases following stimulation, remaining at a significant level for at least 8 h. JunB protein presents the highest expression of all. FosB, like c-Fos, is transiently induced. Pulse-chase experiments show that all of the proteins except JunD are short-lived. We have shown that c-Fos and FosB form complexes in vivo with the different Jun proteins and that JunB complexes are predominant. In vitro association and competition experiments show that the affinities between the different Fos and Jun proteins are similar. This finding, together with the in vivo observations described above, suggests that the proportion of the different Jun/Fos heterodimers is governed by the concentration of the different components. The Fos and Jun proteins are phosphoproteins, and some remain relatively highly phosphorylated in their heterodimeric form.

We have determined the different Fos/Jun complexes present in Swiss 3T3 cells either following serum stimulation of quiescent cells or during exponential growth by immunoprecipitation analyses. We have shown that while c-Fos is the major Fos protein associated with the Jun proteins (c-Jun, JunB, and JunD) soon after serum stimulation, at later times Fra-1 and Fra-2 are the predominant Fos proteins associated with the different Jun proteins. During exponential growth, the synthesis of Fra-1 and Fra-2 is maintained at a significant level, in contrast to c-Fos and FosB, which are expressed at very low or undetectable levels. Consequently, Fra-1 and Fra-2 are the main Fos proteins complexed with the Jun proteins in asynchronously growing cells. To determine whether the Fos proteins are differentially required during the G0-to-G1 transition and exponential growth for the entrance into S phase, we microinjected affinity-purified antibodies directed against c-Fos, FosB, Fra-1, and Fra-2. We have found that while the activities of c-Fos and FosB are required mostly during the G0-to-G1 transition, Fra-1 and Fra-2 are involved both in the G0-to-G1 transition and in asynchronous growth.

Jun, Fos, and Krox proteins are inducible transcription factors contributing to the control of gene expression. The elucidation of their individual expression patterns in the nervous system provides new insights into the ability of neurons to react with changes of gene expression to external stimulation under physiological or pathological conditions. The expression of c-Jun, JunB, JunD, c-Fos, FosB, and Krox-24 was investigated in the brain of untreated male Sprague-Dawley and female BDIX rats by immunocytochemistry using specific antibodies. JunD immunoreactivity (IR) labeled the highest number of neurons, being present in almost all neurons of the brain. JunD was expressed at high levels in those areas that also exhibit c-Jun, JunB, c-Fos, and FosB-IR, such as locus coeruleus, periolivary nuclei (ncl.), pontine and central gray, lateral lemniscal ncl., inferior and superior colliculi, leaflet of geniculate ncl., midline nuclei of thalamus, dorsomedial and paraventricular ncl. of hypothalamus, ncl. supraopticus, dorsolateral part of caudate putamen and lateral septal ncl. In contrast to the high number of JunD-positive neurons, c-Jun, JunB, c-Fos, and FosB proteins were detected in rather low numbers of neurons in these brain areas; the rank of the number of immunopositive neurons was c-Fos > JunB > c-Jun > FosB. Particularly high levels of expression were observed for c-Jun in medullary motoneurons, medial geniculate ncl., arcuate ncl., and dentate gyrus, and for JunB in the CA-1 area of the hippocampus and islands of Calleja. The zinc finger protein Krox-24 was expressed in many neurons of these brain areas, with only discrete Jun- and Fos-IR; additionally, many intensely labeled nuclei were present in spinal ncl. of the trigeminal ventromedial ncl. of the hypothalamus and the CA-1 area of the hippocampus. In the cerebellum, nuclear labeling was detected only for c-Jun, JunD, and Krox-24 in granule cells. JunD-IR was also found in glial cells of gray matter and fiber tracts, whereas glial c-Jun-IR was observed only in fiber tracts. Apart from a weak JunD-IR, some areas did not express Jun, Fos, and Krox proteins such as cuneate and gracile ncl., venterobasal complex of thalamus, globus pallidum, and Purkinje cells of the cerebellum. Our data indicate that inducible transcription factors of the fos, jun, and krox gene families show patterns of individual expression in untreated animals, thereby reflecting different mechanisms and/or thresholds for induction under physiological conditions.

We present the first comparative investigation of the basal and transsynaptically induced expression of c-JUN, JUN B, JUN D, c-FOS, FOS B, and KROX-24 proteins in the spinal cord, using immunocytochemistry with specific antibodies. We demonstrate that electrical stimulation of the sciatic nerve at A delta/C-fiber (not A alpha/beta-fiber) intensity strongly induces the expression of these immediate-early gene-encoded proteins. Basal immunoreactivity was found for c-JUN in motoneurons, for JUN D in almost every cell of the gray matter, and for KROX-24 in the superficial dorsal horn. One hour after electrical stimulation of the sciatic nerve at A delta/C-fiber intensity, expression of all proteins except JUN D reached its maximum. Initially immunoreactivity was restricted to the ipsilateral dorsal horn, but after 4 hours appeared contralaterally. Expression of JUN D was increased only after 4 hours. Within the dorsal horn, the expression of c-JUN, JUN B, FOS B, and KROX-24 was mainly restricted to the superficial layers. Immunoreactivity decreased to basal levels between 8 and 16 hours. c-FOS and JUN D were expressed in both the superficial and deep dorsal horn; in the latter, c-FOS and JUN D persisted longer. Induced JUN D was present the longest and was still visible after 32 hours. In motoneurons of the ipsilateral ventral horn, c-JUN, JUN D, and c-FOS appeared after 8 hours. Surgical exposure of the sciatic nerve evoked a strikingly prolonged expression of all proteins compared to that following electrical stimulation of the sciatic nerve. Our results demonstrate that stimulation of nociceptive A delta- and C-fibers induces early and late expression of proteins encoded by immediate-early genes with a specific temporal and spatial distribution of the expression of each protein. Furthermore, the extent of protein expression reflects the intensity of noxious stimulation.

We demonstrate that a member of thefos family, thefosB gene, gives rise to two transcripts by alternative splicing of exon 4, generating two proteins, FosB of 338 amino acids and a short form, FosB/SF, which contains the DNA binding and dimerization domains but not the 101 amino acids of the C terminus. FosB/SF activates an AP-1-chloramphenicol acetyltransferase construct in NIH 3T3 cells, as determined by transient and stable transfections, although more weakly than does FosB. In contrast to FosB, FosB/SF has lost its ability to repress the dyad symmetry element of the c-fos gene. FosB/SF when expressed in excess to FosB can downmodulate the activity of FosB. Constitutive expression of high levels of FosB/SF in NIH 3T3 cells has no significant inhibitory effect in the induction of cell proliferation or cell cycle progression, indicating that FosB/SF is not a negative regulator of cell growth. This conclusion is further confirmed by the observation that the majority of the Jun molecules are complexed with FosB/SF in the FosB/SF-overexpressing cells.

Naturally occurring antioxidants such as carotenoids are extensively studied for their potential in reducing the risk for cancer and other chronic diseases. In the present study, the radicalscavenger activity of the food additive norbixin, a water-soluble carotenoid extracted from Bixa orellana seeds and commercialized as annatto, was evaluated under conditions of DNA damage induced by reactive oxygen species, particularly by hydroxyl radicals. The cell-free scavenger activity of norbixin was evaluated using plasmid DNA as target molecule and Sn 2+ or Fe 2+ as oxidant. The addition of H 2 O 2 enhanced DNA breakage induced by metal ions, particularly Fe 2+ . Under these conditions, norbixin started to protect plasmid DNA against single-and double-strand breakage at a metal:norbixin ratio of 1:1 (Sn 2+ ) and 1:10 (Fe 2+ ). However, at lower ratios to Sn 2+ , norbixin enhanced Sn 2+ -induced DNA breakage P , 0´05X The ability of norbixin to protect genomic DNA against oxidative damage was assessed in murine fibroblasts submitted to H 2 O 2 -induced oxidative stress and the results were evaluated by the comet assay. Under low serum conditions (2 % fetal bovine serum (FBS)), a protective effect of norbixin against H 2 O 2 -induced DNA breakage was inversely related to its concentration, a protection ranging from 41 % (10 mM) to 21 % (50 mM). At higher concentrations of norbixin, however, oxidative DNA breakage was still enhanced, even in the presence of a high serum concentration (10 % FBS). Under normal conditions, norbixin per se has no detectable genotoxic or cytotoxic effects on murine fibroblasts. The antimutagenic potential of norbixin against oxidative mutagens was also evaluated by the Salmonella typhimurium assay, with a maximum inhibition of 87 % against the mutagenicity induced by H 2 O 2 . Although plasmid DNA and Ames data indicated that norbixin can protect DNA against oxidative damage, it seems to be a risky guardian of genomic DNA as it can also increase the extent of oxidative damage.