Reduced induction of c-fos but not of c-myc expressions in a nontumorigenic revertant R1 of EJ-ras-transformed NIH/3T3 cells treated with 12-O-tetradecanoylphorbol-13-acetate (TPA)

It has been reported that both c-fos and c-myc mRNAs are induced in NIH/3T3 cells after 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment. We have studied the effect of TPA on the expression of c-fos and c-myc in EJ-ras-transformed NIH/3T3 and its nontumorigenic flat revertant R1 cells. Although TPA treatment induces c-myc mRNA, as in the case of NIH/3T3 cells, the induced level of c-fos mRNA is greatly reduced not only in slow-growing EJ-ras-transformed NIH/3T3 but also in quiescent R1 cells. In addition, serum-induced c-fos expression is also reduced in EJ-ras-transformed NIH/3T3 and R1 cells. These observations suggest that the pathway from TPA to c-fos gene is different from that to c-myc gene and that the former pathway is down-regulated in association not with the transformed phenotype, but with EJ-ras expression, and it is possible that this reduced induction of c-fos is not specific to TPA.     

Effect of the Tumor Promoter Phorbol 12-Myristate 13-Acetate (PMA) on Proliferation of Young and Senescent WI-38 Human Diploid Fibroblasts

The effects of the tumor promoter phorbol 12-myristate 13-acetate (PMA) on the proliferation, protein kinase C activity (PKC), and c-fos gene expression were examined in cultures of young and senescent (90-95% lifespan completed) WI-38 human diploid fibroblasts. We observed that, following stimulation with medium containing 10% fetal bovine serum (FBS), the translocation of PKC from the cytosol to the particulate compartment was less efficient in senescent WI-38 cells than in young cells. However, when PMA was added to the medium, the intracellular distribution of PKC activity in old cells became nearly identical to that observed in young cells. The inducibility of c-fos mRNA by serum addition, which is a protein kinase C-dependent event [64], was significantly amplified in the presence of PMA. Moreover, the duration of peak c-fos expression, after stimulation by FBS and PMA, increased in senescent cells as compared to young cells. Our results reveal that the normal signal transduction pathway is altered in senescent, slowly proliferating human fibroblasts and that it can be partially restored in the presence of the tumor promoter PMA.     

The application of antisense oligonucleotide technology to the brain: Some pitfalls

Summary 1. Amphetamine-induced c-fos andegr-1 expression in the striatum was used as a model in which to study the effects of antisense oligodeoxynucleotides (ODNs) directed at c-fos. Using direct infusions of ODNs into the striata of animals we have demonstrated that c-fos antisense ODNs retain most of their biological activity with 2- or 3-base substitutions. The c-fos antisense and mismatch ODNs attenuated Fos immunoreactivity but had little effect on Egr-1 immunoreactivity.2. In another group of studies examining the role of c-fos in amygdala kindling, we have demonstrated that ODNs cause neurotoxic damage following repeated daily infusions into the amygdala. The damage observed was greatly diminished when the time interval between infusions was extended.     

Regulation of c-fos is affected by electromagnetic fields

The goal of the present study was to determine if regulatory regions of the c-fos gene were responsive to electromagnetic field exposure. The research design used transfected cells to increase the sensitivity of assays designed to identify changes following exposure. HeLa cells were transiently transfected with plasmids containing upstream regulating regions of c-fos up to -700 base pairs, coupled with the prokaryotic reporter gene CAT. Cells were exposed to an environmentally relevant EMF of 60 Hz at 60 mG_rms. CAT expression above control levels in transfected cells (region +42 to -700 bp) was observed following 5 min exposure to the electromagnetic field, with a peak at 20 min. The expression was at basal levels following 40 min exposure. Deletion analysis of upstream DNA narrowed the responsive region to 138 base pairs from -363 to -225, which contains the SRE/AP-1 sites. © 1996 Wiley-Liss, Inc.     

Expression of c-fos and jun protooncogenes in ovine trophoblasts in relation to interferon-tau expression and early implantation process

Expression of c-fos and jun protooncogenes was analyzed in the ovine extraembryonic trophoblast from days 14–18 of gestation, using Northern and Western blotting and immunohistochemistry. This study was carried out in relation to the early implantation process and the expression of interferon-tau, which is secreted in large amounts for a few days before implantation. Our results demonstrated that c-fos, c-jun, and junB were differently expressed in the ovine trophoblast around the time of implantation. The c-fos mRNA and protein were detected at high levels prior to attachment and decreased thereafter, following the pattern of expression of interferon-tau, whereas c-jun expression was maintained at relatively high levels during the implantation process. By contrast, the levels of junB mRNA and protein decreased prior to attachment. Immunohistochemical studies indicated that JunB, like C-Fos and interferon tau, was no longer expressed in the trophoblastic cells which had established cellular contacts with the uterine epithelium. A striking finding in this study is the temporal correlation between the accumulation of c-Fos and c-Jun proteins and the expression of the interferon-tau (days 14 and 15 of gestation). We also showed by gel-retardation assays that an AP-1-like site present in the promoter of one interferon-tau gene was functional in vitro, as judged by its ability to bind day-15 trophoblast nuclear protein extracts. Nuclear proteins binding to this site had the characteristics of AP-1, as judged by the ability to be competed efficiently by a consensus TRE (12.0-tetradecanoyl phorbol 13-acetate-responsive element)-site oligonucleotide and by antibodies to c-Fos and Jun proteins. These results suggest that Fos and Jun could form regulatory complexes of interferon-tau expression and/or are regulated by common mechanisms which are still unknown. Mol Reprod Dev 46:127–137, 1997. © 1997 Wiley-Liss, Inc.     

c-fos antisense reduces expression of Krox 24 in rat caudate and neocortex

Summary 1. The aim of this study was to investigate the neurochemical effects and measure the anatomical spread of infusion of c-fos antisense (AS) DNA into the striatum.2. Rats were anesthetized and infused in opposing striata with c-fos AS and c-fos sense (S) DNA. Ten hours later they were injected with apomorphine (2 mg/kg, i.p.) and 20 min later they were overdosed with sodium pentobarbital and their brains either perfused or frozen. Vibratome-cut sections were immunostained for the detection of c-fos, JunB, Krox 24, somatostatin, substance P, dynorphin, tyrosine hydroxylase, and enkephalin. Cryostat-cut sections from the caudate were immunostained for the detection of c-fos, JunB, and Krox 24, as well asin situ hybridization for proenkephalin mRNA. Sections from the globus pallidus were used for the autoradiographic localization of D2 dopamine and A2_a adenosine receptors. Sections from the substantia nigra were used for the autoradiographic localization of D1 dopamine and cannabinoid receptors. A second group of rats was injected in opposing striata with biotin-labeled c-fos AS DNA and c-fos S DNA. Ten hours later they were challenged with apomorphine (2 mg/kg, i.p.) and 20 min later brains were either perfused or frozen. Sections from these brains were cut throughout the rostral-caudal extent of the forebrain and the biotin labeled AS DNA was localized.3. Krox 24 was expressed at high levels on the sense side of the brain in the striatum and overlying neocortex. However, on the AS-injected side there was a reduction in Krox 24 expression in striatum and overlying cortex. The biotin-labeled AS studies confirmed that the striatal infusion spread throughout the dorsal striatum as well as the overlying neocortex. We did not detect any changes in neurotransmitter receptors, neuropeptides, or tyrosine hydroxylase in AS/S-injected rat brains.4. These results demonstrate that c-fos AS reduces Krox 24 expression in striatal and neocortical neurons but does not change the expression of a number of other proteins involved in basal ganglia function. Whether this effect is due to nonspecific actions of c-fos AS or to its effects on a component of the transduction pathway responsible for basal Krox 24 expression (NMDA receptors?) is unknown.