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.     

Expression of c-myc is not critical for cell proliferation in established human leukemia lines

Background

A study was undertaken to resolve preliminary conflicting results on the proliferation of leukemia cells observed with different c-myc antisense oligonucleotides.

Results

RNase H-active, chimeric methylphosphonodiester / phosphodiester antisense oligodeoxynucleotides targeting bases 1147–1166 of c-myc mRNA downregulated c-Myc protein and induced apoptosis and cell cycle arrest respectively in cultures of MOLT-4 and KYO1 human leukemia cells. In contrast, an RNase H-inactive, morpholino antisense oligonucleotide analogue 28-mer, simultaneously targeting the exon 2 splice acceptor site and initiation codon, reduced c-Myc protein to barely detectable levels but did not affect cell proliferation in these or other leukemia lines. The RNase H-active oligodeoxynucleotide 20-mers contained the phosphodiester linked motif CGTTG, which as an apoptosis inducing CpG oligodeoxynucleotide 5-mer of sequence type CGNNN (N = A, G, C, or T) had potent activity against MOLT-4 cells. The 5-mer mimicked the antiproliferative effects of the 20-mer in the absence of any antisense activity against c-myc mRNA, while the latter still reduced expression of c-myc in a subline of MOLT-4 cells that had been selected for resistance to CGTTA, but in this case the oligodeoxynucleotide failed to induce apoptosis or cell cycle arrest.

Conclusions

We conclude that the biological activity of the chimeric c-myc antisense 20-mers resulted from a non-antisense mechanism related to the CGTTG motif contained within the sequence, and not through downregulation of c-myc. Although the oncogene may have been implicated in the etiology of the original leukemias, expression of c-myc is apparently no longer required to sustain continuous cell proliferation in these culture lines.     

c-fos expression in the amygdala:In vivo antisense modulation and role in anxiety

Summary 1. The amygdaloid complex is a key structure in mechanisms of fear and anxiety. Expression of the immediate-early gene c-fos has been reported in the central nucleus of the amygdala following various stressors, but the functional role of this phenomenon has remained unknown.2. c-fos expression was observed in the central nucleus when rats were subjected to a pharmacologically validated animal model of anxiety, the Vogel conflict test, but not after mere exposure to the test apparatus. Bilateral amygdala injection of a 15-mer phosphorothioate c-fos antisense oligodeoxynucleotide prior to testing blocked conflict-induced c-fos expression and had behavioral effects similar to those of established antianxiety drugs.3. Separate experiments determined that antisense treatment did not affect conflict behavior by acting on shock thresholds or drinking motivation.4. These findings provide evidence that neuronal activation and c-fos induction in the amygdala may be of importance for mechanisms of fear and anxiety.     

Expression of c-fos and c-myc protooncogenes in an immortalized hepatocyte line harbouring SV40 T antigen and hGH as transgenes

A clonal hepatocyte line (FMH-202-2), derived from livers of fetal transgenic mice harbouring human growth hormone (hGH) and SV40 T antigen as transgenes, was used in the investigation of protooncogene expression involved in liver-specific growth control and/or in hepatocellular transformation. In this model system, representing an immortalized, yet untransformed phenotype, the transgenes hGH and SV40 T antigen were expressed constitutively. The c-fos protooncogene was induced by incubation with insulin, epidermal growth factor (EGF) and insulin-like growth factor (IGF-I) in a transient manner comparable to its expression in primary murine hepatocytes. Elucidation of second messenger mechanisms demonstrated that c-fos induction by hepatotrophic growth factors was not mediated by protein kinase C. In contrast to primary hepatocytes, the c-myc protooncogene exhibited a constitutive expression pattern which was independent of growth factor stimulation. These results indicate that apart from hGH and SV40 T antigen, c-myc may play a role in cellular immortalization, but that constitutive expression of these genes, even in combined coexpression, does not suffice to induce the transformed phenotype.     

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.     

Anti-c-myc DNA increases differentiation and decreases colony formation by HL-60 cells

Summary The proto-oncogene c-myc, whose gene product has a role in replication, is overexpressed in the human promyelocytic leukemia HL-60 cell line. Treatment of HL-60 cells with an antisense oligodeoxyribonucleotide complementary to the start codon and the next four codons of c-myc mRNA has previously been observed to inhibit c-myc protein expression and cell proliferation in a sequence-specific, dose-dependent manner. Comparable effects are seen upon treatment of HL-60 cells with dimethylsulfoxide (Me₂SO), which is also know to induce granulocytic differentiation of HL-60 cells. Hence, the effects of antisense oligomers on cellular differentiation were examine and compared with Me₂SO. Differentiation of HL-60 cells into forms with granulocytic characteristics was found to be enhanced in a sequence-specific manner by the anti-c-myc oligomer. No synergism was observed between the anti-c-myc oligomer and Me₂SO in stimulating cellular differentiation. In contrast, synergism did appear in the inhibition of cell proliferation. Finally, the anti-c-myc oligomer uniformly inhibited colony formation in semisolid medium. It is possible that further reduction in the level of c-myc expression by antisense oligomer inhibition may be sufficient to allow terminal granulocytic differentiation and reverse transformation. This work was supported by grants to E. W. from the National Institutes of Health, Bethesda, MD (CA 42960), and the Leukemia Society of America.     

Decreases in local hormone biosynthesis and c-fos gene expression accompany differentiation of porcine preadipocytes

Summary To better understand possible autocrine or paracrine mechanisms involved in adipose tissue development, we have studied the biosynthesis of insulinlike growth factor I (IGF-I) and prostaglandin E₂ (PGE₂) by cultured porcine preadipocytes in response to factors known to modulate cell growth and differentiation. The expression of c-fos was also monitored because of the potential role of that proto-oncogene in coordination of growth and differentiation. Preadipocytes were grown to confluence and then maintained in one of three media treatments: a) standard medium supplemented with 10% fetal bovine serum (FBS), b) FBS supplemented with dexamethasone (Dex), c) FBS supplemented with dibutryladenosine 3′–5′-cyclic monophosphate. Indirect measurements of growth indicated that cell proliferation did not differ due to media type. Histochemical and enzymatic measurements of adipocyte development revealed that differentiation occurred only in those cultures exposed to Dex. The increase in adipocyte differentiation in response to Dex was associated with a decrease in c-fos and actin RNA expression whereas the decrease in c-fos RNA expression in response to Dex was small (approximately 40%); immunocytochemical analysis indicated that induction of Fos protein occurred only in undifferentiated cells. Thus, the cells responsible for the decrease in c-fos RNA expression are possibly those signaled to differentiate into adipocytes. Expression of IGF-I RNA and secretion of IGF-I and PGE₂ were also decreased in response to Dex treatment. These data provide the first demonstration that biosynthesis of IGF-I by preadipocytes can be modulated by a potent inducer of adipocyte differentiation. The combined results indicate that glucocorticoids may stimulate adipocyte differentiation by suppressing intracellular and putative intercellular mitogenic signals. This work was supported in part by grant HD 18447 from the National Institutes of Health, Bethesda, MD (G. J. H.). Mention of a trade mark, proprietary product, or specific equipment does not constitute a guarantee or warranty by the U. S. Department of Agriculture or University of Georgia and does not imply its approval to the exclusion of other products that may be suitable.     

Purification and characterization of interferon-like antiviral protein derived from flatfish (Paralichthys olivaceus) lymphocytes immortalized by oncogenes

Flatfish leukocytes were transfected with the expression plasmids of the v-myc, c-myc, c-fos, v-myb and c-Ha-ras oncogenes. Only cotransfection of c-Ha-ras with c-myc or c-fos resulted in complete immortalization of the cells. Interferon-like anti-viral protein was found in the cultured medium of the immortalized lymphocytes. The protein was purified by DEAE-Toyopearl 650 M ion exchange chromatography and WGA agarose affinity chromatography. The protein was a glycoprotein of about 16 kDa. The antiviral activity of the protein was trypsin-sensitive and was fairly stable at pH values from 4 to 8. The protein retained about 60% of the activity even at 60°C and showed a broad antiviral activity for various fish cells and viruses.     

Docosahexaenoic acid-containing phosphatidylethanolamine enhances HL-60 cell differentiation by regulation of c-jun and c-myc expression

18:1/docosahexaenoic acid (DHA)-containing phosphatidylethanolamine (PE) enhanced cell differentiation and growth inhibition of HL-60 induced by dibutyryl cAMP (dbcAMP) in a dose-dependent manner. The combined treatment of 200 μM dbcAMP and 50 μM 18:1/DHA-PE increased the NBT reducing activity, which is as an indicator of cell differentiation, to more than 75% from 40% of cells treated with 200 μM dbcAMP alone. In HL-60 cells treated with 50 μM 18:1/DHA-PE and 200 μM dbcAMP for 24 h, the expression level of c-jun mRNA and c-Jun protein were remarkably elevated compared to cells treated with dbcAMP alone. In contrast, there was no difference in the expression levels of c-fos mRNA and c-Fos protein between the combination of 18:1/DHA-PE + dbcAMP or dbcAMP alone. On the other hand, the combine treatment of 18:1/DHA-PE and dbcAMP markedly reduced the expression level of c-myc oncogene during 48 h incubation. The decreases of c-myc mRNA by 18:1/DHA-PE and/or dbcAMP was correlated with growth inhibition effect. Thus, 18:1/DHA-PE might enhance dbcAMP-induced HL-60 cell differentiation and growth inhibition by regulation of c-jun and c-myc mRNA and their products.