Chronic Electroconvulsive Seizures Down–Regulate Expression of the Immediate-Early Genes c-fos and c-jun in Rat Cerebral Cortex

Acute seizures and other stimuli that increase neuronal activity cause a rapid induction of the immediate-early genes c-fos and c-jun, also referred to as nuclear proto-oncogenes, in the nervous system. In the present study, rats were administered one or more electroconvulsive seizures (ECS) and the responsiveness of c-fos and c-jun to an acute, "test" seizure was examined. Four hours after a single ECS, the induction of c-fos mRNA by a test seizure was blocked, in agreement with earlier findings, but by 18 h the levels of c-fos mRNA could be reinduced by the test seizure, suggesting that 1 day is sufficient to "reset" the responsiveness of this system. However, it was found that chronic, daily ECS treatments resulted in a time-dependent decrease in the expression of c-fos mRNA in response to a test seizure administered 18 h after the last daily ECS; this effect was maximal after 8-10 days of treatment, at which time the induction of c-fos mRNA by the test seizure was blocked dramatically. Chronic ECS also blocked the induction of c-jun in response to an acute, test seizure. The effect of chronic ECS on levels of Fos protein was also investigated. It was found that basal levels of Fos protein were reduced after chronic (10 days) ECS and were not induced by a test seizure. Because levels of Fos protein remain elevated 4 h after a single seizure this finding suggests that the mechanisms by which acute (4 h) and chronic (8-10 days) ECS block the induction of c-fos may differ.     

Hydrogen peroxide induces heat shock protein and proto-oncogene mRNA accumulation in Xenopus laevis A6 kidney epithelial cells

In this study, we examined the effect of hydrogen peroxide on the accumulation of various mRNAs encoding heat shock proteins (hsps) and proto-oncogenes in Xenopus A6 kidney epithelial cells. Hydrogen peroxide treatment enhanced the accumulation of hsp90, hsp70, hsp30, c-jun, c-fos, and actin mRNAs with distinct temporal patterns. Although hsp70, c-fos, and c-jun mRNA levels peaked at 1-2 h before declining, hsp30 and hsp90 mRNA levels were maximal at 4-6 h. Other mRNAs, including heat shock cognate hsc70, immunoglobulin binding protein, and ribosomal L8, were unaffected. Treatment of kidney cells with a combination of mild heat shock plus hydrogen peroxide resulted in a synergistic increase in the relative levels of both hsp70 and hsp30 mRNA, but not hsp90, c-fos, c-jun, or actin. This study suggests that analysis of hsp and proto-oncogene mRNA levels may be of value as molecular biomarkers of oxidative stress associated with various disease states and nephrotoxicity in kidney.     

Regulation of proto-oncogenes in rat parotid acinar cells in vitro after stimulation of beta-adrenergic receptors

Stimulation of beta-adrenoreceptors in rat parotid acinar cells in vitro by the beta-adrenergic agonist isoproterenol induces steady-state levels of c-fos mRNA and c-fos protein in these cells. A dramatic increase in the steady-state levels of c-fos mRNA was observed at 60 min, followed by a decrease at 2 h with a second peak at 4 h. c-fos induction in rat parotid acinar cells in vitro seems to be mediated by cAMP. Increased levels of p53 and c-myc mRNA were detected only at 60 min. c-abl and c-sis were also induced by isoproterenol but in a pattern different from that seen with c-fos. c-abl was the only oncogene in rat parotid gland which showed increased expression after chronic isoproterenol treatment of rats. In rat parotid acinar cells we observed no correlation between DNA synthesis and c-fos induction.     

c-fos and c-myc expression in human endothelial cells as a function of different culture conditions

Cultured human umbilical vein endothelial cells (HEC) could be induced to express c-fos and c-myc mRNA by either serum or ECGS (endothelial cell growth supplement). Neither agonist separately could support HEC proliferation but the combination did. Expression of c-fos and c-myc mRNA in the presence of both serum and ECGS was similar to that observed after each of the two stimuli was introduced separately. c-fos and c-myc expression in cultured HEC, even if related, is not necessarily accompanied by stimulation of cell growth.     

The effect of LPS on expression of the early "competence" genes JE and KC in murine peritoneal macrophages

The expression of early "competence" genes has been examined in murine peritoneal macrophages treated with bacterial lipopolysaccharide (LPS). This set of genes (e.g., c-myc, c-fos, r-fos, JE, and KC) were first described in BALB/c 3T3 cells treated with platelet-derived growth factor. We have previously reported that LPS induces the rapid and transient expression of both c-myc and c-fos in macrophages. In the present report, we present evidence demonstrating that the mRNA for JE and KC are also induced in macrophages after treatment of LPS. The r-fos gene was not detectably induced by LPS under the experimental conditions used in this study. The induction of JE and KC were dependent upon the dose of LPS and exhibited different time courses. mRNA for both KC and JE was induced within 30 min from the initiation of treatment. Although mRNA for JE continued to accumulate for up to 24 hr, mRNA for KC was optimally seen after 60 min and had disappeared by 4 hr. c-fos, JE, and KC mRNA were all inducible by a variety of structurally diverse but functionally similar agents (e.g., heat killed Listeria monocytogenes, maleyl-bovine serum albumin, and fucoidan). Interferon-gamma, a potent but functionally distinct stimulus of macrophage activation, did not effect the expression of JE or KC mRNA. The expression of mRNA for c-fos could be readily induced by treatment of macrophages with phorbol myristate acetate (PMA) alone and that for JE by PMA plus the inophore A23187; mRNA for KC was largely unaffected by these agents. These results suggest that expression of the c-fos and JE genes are regulated by products of polyphosphoinositide hydrolysis. The difference between c-fos or JE and KC raises the possibility that LPS may stimulate at least two independent routes of early gene expression. LPS does not promote macrophage proliferative activity alone, and in fact inhibits the proliferative response to the macrophage growth factor colony-stimulating factor 1. Taken together these findings suggest that the products of these genes may function in the acquisition of competence for highly differentiated functions in addition to that for cell division.     

Time of c-fos and c-myc expression in human diploid fibroblasts stimulated to proliferate after prolonged periods in quiescence

When human diploid fibroblasts such as WI-38 cells become crowded, they enter a viable state of quiescence (G0) in which they can remain for prolonged periods of time. These quiescent cells can be induced to re-enter the cell cycle by addition of fresh serum. However, cells held in G0 for long periods before stimulation require more time to enter DNA synthesis as compared to cells held in a quiescent state for short periods. We have used this model system to determine if a close temporal coupling exists between the time of expression of two proto-oncogenes associated with cell growth, c-fos and c-myc, and the time of entry into DNA synthesis. WI-38 cells were stimulated to enter DNA synthesis by the addition of fresh culture medium and serum at various lengths of time after plating, ranging from 7 to 34 days. At hourly intervals thereafter, cells were harvested and total RNA was isolated. These samples were then analyzed by RNase protection assay to determine the levels of c-fos and c-myc mRNA. Our results show that the time and pattern of c-fos and c-myc mRNA accumulation after stimulation is determined only by the time which the cells are treated with serum even when they exhibit a 19-h delay in the entry into DNA synthesis. In all of our experiments, c-fos could be detected 0.5 h after stimulation and remained detectable for approximately 2 h. Likewise, the peak of c-myc accumulation occurred at about 3 h after serum addition, regardless of how long it took to initiate DNA synthesis. These results suggest that the time of c-fos and c-myc induction clearly is not the only factor which determines the length of the prereplicative period and thus the ultimate time of initiation of DNA synthesis.     

Tumor necrosis factor and interleukin-1 cause a rapid and transient stimulation of c-fos and c-myc mRNA levels in human fibroblasts

Tumor necrosis factor (TNF) and interleukin-1 (IL-1) were shown previously to be mitogenic for human fibroblasts. Here we show that recombinant human TNF and recombinant human IL-1 alpha increase steady state levels of c-fos and c-myc proto-oncogene mRNAs in quiescent human FS-4 fibroblasts. Proto-oncogene mRNA levels were enhanced within 20 min of TNF or IL-1 addition, peaked at 30 min, and declined to undetectable levels (c-fos) or basal levels (c-myc) by 60 or 90 min. A similar rapid increase in c-fos and c-myc mRNA was seen in quiescent FS-4 cells exposed to cycloheximide. However, in the presence of cycloheximide, both proto-oncogene mRNA levels continued to rise for at least 90 min. The transient nature of the increase in c-myc mRNA levels appears to be a response characteristic for TNF and IL-1 because in quiescent FS-4 cells exposed to 10% fetal bovine serum, steady state levels of c-myc mRNA remained elevated for at least 4 h.     

Expression of c-fos oncogene during hepatocarcinogenesis, liver regeneration and in synchronized HTC cells

We have studied the expression of c-fos gene in rat hepatoma induced by DENA. An increase of c-fos mRNA concentration was observed after 8 days, but the maximal 5- to 6-fold increase was observed after 70 weeks. This increase was found in perinodular hepatocytes as well as in cancer nodules. c-fos expression was also enhanced during liver regeneration at a period corresponding to cell proliferation. In HTC cells the arrest of the cell cycle at early G1 phase by addition of sodium butyrate was accompanied by a strong increase of c-fos gene expression. However the c-fos mRNA rapidly decreased after removal of sodium butyrate during the progression of the cells in the cell cycle and increased transiently when the cells entered again in G1 phase.     

Regulation of proliferation in a murine colony-stimulating factor-dependent myeloid cell line: superinduction of c-fos by the growth inhibitor 8-Br-cyclic adenosine 3':5' monophosphate

We have investigated the effect of 8-Br-cyclic adenosine 3':5' monophosphate (cAMP), a pharmacological activator of cAMP-dependent protein kinase, on the proliferation and the nuclear proto-oncogene induction in a murine granulocyte macrophage colony-stimulating factor (GM-CSF)-dependent myeloid cell line. Cells were growth arrested by granulocyte macrophage colony-stimulating factor and serum deprivation and were allowed to proceed in the cell cycle by addition of the lymphokine in the presence or absence of 8-Br-cAMP. 3H-thymidine incorporation assays showed that addition of 8-Br-cAMP inhibited the entry of cells into S phase and the subsequent proliferation. Northern analysis showed that 8-Br-cAMP had opposite effects on c-fos and c-myc mRNA induction. 8-Br-cAMP induced c-fos in the absence of any GM-CSF. In the presence of GM-CSF, c-fos mRNA was superinduced (30-fold induction compared to four- to fivefold by each signal alone). On the contrary, 8-Br-cAMP was not able to induce c-myc in the absence of growth factor and hardly interfered with the induction of c-myc by GM-CSF. Phorbol myristate acetate (PMA), a pharmacological activator of the lipid and CA++-dependent protein kinase C, was shown to induce nuclear proto-oncogene mRNA in the GM-CSF-dependent cell line. We investigated the effect of 8-Br-cAMP on PMA-induced c-fos and c-myc mRNA levels. When both cAMP dependent and lipid-dependent kinase systems were co-stimulated in the absence of GM-CSF, c-fos message was again superinduced (60-fold induction). On the contrary, c-myc message induction by PMA was inhibited by 80% by coactivation of cAMP-dependent protein kinase with 8-Br-cAMP. Our data indicate that an antiproliferative signal induces or even superinduces c-fos message and hardly interferes with c-myc induction, suggesting that the intracellular pathways resulting in c-fos and c-myc induction may be distinct and that two different pathways can lead to c-fos induction, with synergistic effects when both are activated.     

Variations in c-fos mRNA expression during serum induction and the synchronous cell cycle

Induction of c-fos mRNA levels associated with the stimulation of growth by fetal bovine serum following quiescence was examined in three cell types following brief (24 h) serum starvation. Starved NIH-3T3 and HeLa S3 cells experienced c-fos mRNA induction 20-30 min after addition of serum. In contrast, Swiss-3T3 cells expressed c-fos constitutively following serum starvation. The pattern of oncogene expression coincided with the level of quiescence of each cell line prior to induction. Serum inductions of c-fos expression was dependent upon the response of each cell line to serum starvation, c-fos expression was also examined in HeLa S3 cells that had been separated into sequential cell cycle phases by centrifugal elutriation, c-fos expression peaked during the earliest part of the synchronous G1 phase. The amount of c-fos mRNA measured was approximately twice that found during other cell cycle phases. This suggests that, in addition to its role during the transition from quiescence, the c-fos gene product may play a regulatory role during the earliest part of G1 phase of the continuous cell cycle.