Epidermal growth factor induces tyrosine hydroxylase in a clonal pheochromocytoma cell line, PC-G2.

We have previously described the isolation of a clonal cell line (PC-G2) in which the level of tyrosine hydroxylase (TH), the rate-limiting step in the synthesis of the catecholamine neurotransmitters, is induced by nerve growth factor (NGF). We now report that epidermal growth factor (EGF) also induces TH in the PC-G2 cell line. Although EGF has been shown to be mitogenic for many cultured cells, no neuronal function has been previously reported for this protein. The TH response to EGF is elicited in a dose-dependent fashion at concentrations as low as 0.1 ng/ml and is maximal at 10 ng/ml EGF. The maximal response is observed after 3--4 d of exposure to 10 ng/ml EGF. The induction by NGF and EGF is inhibited by their respective antisera. Dexamethasone, a synthetic glucocorticoid which we have previously shown modulates the response of PC-G2 cells to NGF, also modulates the TH induction elicited by EGF.

Dexamethasone stimulation of metallothionein synthesis in HeLa cell cultures.

HeLa cells in culture synthesize metallothionein. To investigate the effects of glucocorticoids on metallothionein synthesis we adapted HeLa cells to growth in a defined medium lacking hydrocortisone. In this defined medium, containing 1.5 x 10(-6)M Zn2+, dexamethasone (10(-6)M) caused a five- to sixfold increase in the synthesis of metallothionein. Progesterone (10(-5)M), a known antagonist of glucocorticoids, inhibited this response by 50 percent.

Ultraviolet irradiation transforms C3H10T1/2 cells to a unique, suppressible phenotype.

Transformation of C3H10T1/2 cells by exposure to ultraviolet (UV) irradiation followed by tetradecanoyl phorbol acetate (TPA) has been used as a model of two-stage carcinogenesis. However, cells cloned from UV-TPA-induced foci (UV-TDTx cells) had a unique phenotype. Cloned UV-TDTx cells appeared transformed in pure culture but were unable to form foci when cocultured with C3H10T1/2 cells. However, in the presence of TPA, UV-TDTx cells form foci in mixed culture with C3H10T1/2 cells. This phenotype was the only one observed for UV-TPA transformants. These data suggest that communal suppression of cell division is a discrete phenomenon that must be overcome as one step in the multistage process of transformation, and this protocol permits the routine isolation of transformed cells responsive to density-dependent growth suppression.

S-100 protein synthesis by isolated polyribosomes from rat brain.

The radioactive proteins synthesized in a cell-free system and released from polyribosomes from rat brain were analyzed for the presence of a protein found only in the nervous system. Polyribosomes from rat liver were also studied to demonstrate the organ specificity of the radioactive product synthesized in vitro. The S-100 protein constituted 0.15 percent of the radioactive proteins released from brain polyribosomes.

Extracellular signals, transcriptional responses and cellular specificity.

The expression of a relatively small set of common primary response genes is frequently induced, in a variety of cellular responses, by growth factors, protein hormones and neurotransmitters. The characterization of these genes and their products may provide clues to the mechanisms by which ligand- and cell-specific responses may be generated.

Transcriptional induction of prostaglandin G/H synthase-2 by basic fibroblast growth factor.

In serum-free mouse osteoblastic MC3T3-E1 cells, basic fibroblastic growth factor (bFGF) induced mRNA and protein for prostaglandin G/H synthase-2 (PGHS-2), the major enzyme in arachidonic acid (AA) conversion to prostaglandins. mRNA accumulation peaked at 1 h with bFGF 1 nM. In cells stably transfected with a 371-bp PGHS-2 promoter-luciferase reporter, bFGF stimulated luciferase activity, which peaked at 2-3 h with bFGF 1-10 nM. In the presence of exogenous AA, bFGF stimulated PGE2 production, which paralleled luciferase activity. In serum-free neonatal mouse calvarial cultures, bFGF stimulated PGE2 production in the absence of exogenous AA. bFGF stimulated PGHS-2 mRNA accumulation, which peaked at 2-4 h and then decreased; there were later mRNA elevations at 48 and 96 h that were inhibited by indomethacin. In both MC3T3-E1 cells and neonatal calvariae, bFGF produced smaller and slower increases in PGHS-1 mRNA levels than for PGHS-2. bFGF stimulated bone resorption in mouse calvariae with a maximal increase of 80% at 1 nM. Stimulation was partially inhibited by nonsteroidal anti-inflammatory drugs. We conclude that bFGF rapidly stimulates PGE2 production in osteoblasts, largely through transcriptional regulation of PGHS-2, and that prostaglandins mediate some of bFGF's effects on bone resorption.

A 12-O-tetradecanoylphorbol-13-acetate (TPA)-nonproliferative variant of 3T3 cells is resistant to TPA-enhanced gene amplification.

Barsoum and Varshavsky (Proc. Natl. Acad. Sci. U.S.A. 80:5330-5334, 1983) suggest that polypeptide mitogens and the mitogenic tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) stimulate gene amplification by related pathways. I demonstrated that TPA and the polypeptide mitogen fibroblast growth factor (FGF) both increase the frequency of cadmium-resistant variants of Swiss-Webster 3T3 cells. The molecular basis for this phenomenon is the amplification of the metallothionein gene(s). To further characterize the relationship between mitogenesis and gene amplification, I examined the ability of TPA and FGF to increase the frequency of cadmium-resistant colonies in the 3T3 variant cell line 3T3-TNR9. Unlike 3T3 cells, 3T3-TNR9 cells cannot be stimulated by TPA to divide (E. Butler-Gralla and H. R. Herschman, J. Cell. Physiol. 107:59-68, 1981). TPA does not induce an increase in cadmium-resistant colonies of the TPA-nonproliferative 3T3-TNR9, variant, in contrast to its efficacy on 3T3 cells. FGF, a potent mitogen for 3T3-TNR9 cells as well as 3T3 cells, is equally effective for 3T3-TNR9 and 3T3 cells in inducing cadmium-resistant colonies. These data suggest that the pathways of TPA-induced gene amplification and TPA-stimulated mitogenesis share a common step(s). TPA caused transient inhibition of DNA synthesis in both dividing 3T3 and 3T3-TNR9 cells, suggesting that this latter response to TPA is not sufficient to enhance gene amplification.

Alternative inducers of the rat metallothionein I gene cause distinct changes in chromatin structure in the 5' region of the gene.

We examined the chromatin structure of the rat metallothionein I gene, both in uninduced cells and in cells induced by heavy metals or dexamethasone, using hypersensitivity to DNase I as an assay. The metallothionein I gene of the H4IIE rat hepatoma cell line, expressed at basal level, has a single DNase I-hypersensitive site. This site maps between putative hormone and basal level control sequences. Induction of the gene by cadmium or zinc resulted in the appearance of a new hypersensitive site near the start site of transcription, in a region near the metal-regulatory elements. In contrast, induction of the metallothionein I gene by dexamethasone did not alter the basal pattern of hypersensitivity. Thus, different mechanisms of induction of metallothionein transcription lead to distinct alterations in the chromatin containing the 5' sequences regulating the expression of this gene.

Butyrate-induced changes in nuclease sensitivity of chromatin cannot be correlated with transcriptional activation.

We examined in the H4IIE rat hepatoma cell line the relationship between butyrate-induced changes in the nuclease sensitivity of chromatin and changes in transcriptional activity of specific genes. The butyrate-inducible metallothionein I (MT-I) gene underwent a dramatic increase in DNase I sensitivity after 3 h of butyrate treatment. However, genes not transcribed in H4IIE cells underwent the same changes in DNase I sensitivity. Thus, butyrate-induced increases in DNase I sensitivity are not sufficient for the transcriptional activation of a gene. Butyrate treatment has also been reported to alter the sensitivity of sequences to micrococcal nuclease (MNase) in a manner reflecting their tissue-specific expression. Butyrate exposure caused increased digestion of the MT-I gene by MNase. However, butyrate-induced MNase sensitivity also occurred for genes which are neither transcribed in untreated cells nor butyrate inducible. Moreover, cadmium, a potent transcriptional activator of the MT-I gene, does not alter the sensitivity of the MT-I gene to MNase. Thus, the butyrate-induced alterations in MNase sensitivity are neither sufficient for, necessary for, nor indicative of transcriptional activation.

Rat metallothionein-1 structural gene and three pseudogenes, one of which contains 5'-regulatory sequences.

As shown by Southern blot analysis, the metallothionein-1 (MT-1) genes in rats comprise a multigene family. We present the sequence of the MT-1 structural gene and compare its features with other metallothionein genes. Three MT-1 pseudogenes which we sequenced apparently arose by reverse transcription of processed mRNA transcripts. Two of these, MT-1 psi a and MT-1 psi c, are retrogenes which derive from the MT-1 mRNA, having diverged from the MT-1 gene 6.9 and 2.6 million years ago, respectively. The third, MT-1 psi b, differs from the MT-1 cDNA by only three nucleotide alterations. Surprisingly, MT-1 psi b also preserves sequence homology for 142 base pairs 5' to the transcription initiation site of the parent gene; it contains a promoter sequence sufficient for specifying metal ion induction. We identified, by S1 nuclease mapping, an RNA polymerase II initiation site 432 base pairs 5' of the MT-1 transcription initiation site of the MT-1 structural gene which could explain the formation of the mRNA precursor to this pseudogene. We were unable to detect MT-1 psi b transcripts, either in liver tissue or after transfection. We conclude that the absence of detectable transcripts from this pseudogene is due to either a reduced level of transcription or the formation of unstable transcripts as a consequence of the lack of a consensus sequence normally found 3' of transcription termination in the MT-1 structural gene.

Induction of tumor promotor-inducible genes in murine 3T3 cell lines and tetradecanoyl phorbol acetate-nonproliferative 3T3 variants can occur through protein kinase C-dependent and -independent pathways.

We isolated a group of genes that are rapidly and transiently induced in 3T3 cells by tetradecanoyl phorbol acetate (TPA). These genes are called TIS genes (for TPA-inducible sequences). Epidermal growth factor (EGF), fibroblast growth factor (FGF), and TPA activated TIS gene expression with similar induction kinetics. TPA pretreatment to deplete protein kinase C activity did not abolish the subsequent induction of TIS gene expression by epidermal growth factor or fibroblast growth factor; both peptide mitogens can activate TIS genes through a protein kinase C-independent pathway(s). We also analyzed TIS gene expression in three TPA-nonproliferative variants (3T3-TNR2, 3T3-TNR9, and A31T6E12A). The results indicate that (i) modulation of a TPA-responsive sodium-potassium-chloride transport system is not necessary for TIS gene induction either by TPA or by other mitogens and (ii) TIS gene induction is not sufficient to guarantee a proliferative response to mitogenic stimulation.

Tumor promoter-inducible genes are differentially expressed in the developing mouse.

TIS genes are rapidly and transiently induced by tetradecanoyl phorbol acetate in 3T3 cells. We analyzed the developmental appearance of a number of the TIS genes to determine whether, in a normal physiological context, these genes have common or distinct mechanisms of regulation. Each TIS gene has a distinct tissue specificity and/or developmental profile.

v-src induction of the TIS10/PGS2 prostaglandin synthase gene is mediated by an ATF/CRE transcription response element.

We recently reported the cloning of a mitogen-inducible prostaglandin synthase gene, TIS10/PGS2. In addition to growth factors and tumor promoters, the v-src oncogene induces TIS10/PGS2 expression in 3T3 cells. Deletion analysis, using luciferase reporters, identifies a region between -80 and -40 nucleotides 5' of the TIS10/PGS2 transcription start site that mediates pp60v-src induction in 3T3 cells. This region contains the sequence CGTCACGTG, which includes overlapping ATF/CRE (CGTCA) and E-box (CACGTG) sequences. Gel shift-oligonucleotide competition experiments with nuclear extracts from cells stably transfected with a temperature-sensitive v-src gene demonstrate that the CGTCACGTG sequence can bind proteins at both the ATF/CRE and E-box sequences. Dominant-negative CREB and Myc proteins that bind DNA, but do not transactivate, block v-src induction of a luciferase reporter driven by the first 80 nucleotides of the TIS10/PGS2 promoter. Mutational analysis distinguishes which TIS10/PGS2 cis-acting element mediates pp60v-src induction. E-box mutation has no effect on the fold induction in response to pp60v-src. In contrast, ATF/CRE mutation attenuates the pp60v-src response. Antibody supershift and methylation interference experiments demonstrate that CREB and at least one other ATF transcription factor in these extracts bind to the TIS10/PGS2 ATF/CRE element. Expression of a dominant-negative ras gene also blocks TIS10/PGS2 induction by v-src. Our data suggest that Ras mediates pp60v-src activation of an ATF transcription factor, leading to induced TIS10/PGS2 expression via the ATF/CRE element of the TIS10/PGS2 promoter. This is the first description of v-src activation of gene expression via an ATF/CRE element.

The TIS11 primary response gene is a member of a gene family that encodes proteins with a highly conserved sequence containing an unusual Cys-His repeat.

The TIS11 primary response gene is rapidly and transiently induced by both 12-O-tetradecanoylphorbol-13-acetate and growth factors. The predicted TIS11 protein contains a 6-amino-acid repeat, YKTELC. We cloned two additional cDNAs, TIS11b and TIS11d, that contain the YKTELC sequence. TIS11, TIS11b, and TIS11d proteins share a 67-amino-acid region of sequence similarity that includes the YKTELC repeat and two cysteine-histidine containing repeats. TIS11 gene family members are not coordinately expressed: (i) unlike TIS11, the TIS11b and TIS11d mRNAs are detectable in quiescent Swiss 3T3 cells and are not dramatically induced by 12-O-tetradecanoylphorbol-13-acetate; (ii) cycloheximide superinduction does not occur for TIS11b and TIS11d; and (iii) unlike TIS11, TIS11b expression is extinguished in PC12 pheochromocytoma cells.

Metal-dependent binding of a factor in vivo to the metal-responsive elements of the metallothionein 1 gene promoter.

Using the technique of genomic footprinting, we demonstrate cadmium-inducible protection from dimethyl sulfate (DMS) modification of guanine residues in vivo in five metal-responsive elements (MREs) in the promoter of the rat metallothionein 1 (MT-1) gene. We also identify a site of extreme DMS hyperreactivity which, like the MRE protection, occurs only after metal ion induction. With this hyperreactive site as an indicator, we can measure the kinetics of induction and deinduction. Changes in the intracellular metal ion concentrations are reflected in alterations in the reactivity with DMS of guanine residues in the MT-1 gene promoter. Lastly, for both control and metal-induced cells, we observe DMS protection and enhancement of a binding site (located 5' of the distal MRE) which is a consensus sequence for the Sp1 transcription factor. Transfection experiments with deletion mutations of a fusion gene construct indicate both that a sequence region which includes this GC box regulates the basal level of expression of the MT-1 gene and that increasing the number of MREs in the promoter increases the induced level of transcription. Our genomic footprinting and transfection data together suggest that (i) a transcription factor, possibly Sp1, plays an important role in regulating the basal level of expression of the MT-1 gene and (ii) metal induction involves the metal-dependent binding to a sequence-specific binding factor which responds to changes in intracellular metal ion levels.

Protein phosphorylation in a tetradecanoyl phorbol acetate-nonproliferative variant of 3T3 cells.

The 3T3-TNR9 cell line is a variant of Swiss 3T3 cells which does not respond mitogenically to tumor promoters, but does respond mitogenically to epidermal growth factor, fibroblast growth factor, and serum. To elucidate differences between tumor promoters and polypeptide mitogens in the pathway(s) of mitogenesis which might be responsible for the nonresponsiveness of the 3T3-TNR9 cells, we have examined in these cells the early protein phosphorylation events known to be associated with mitogenesis in the parental 3T3 cells. We find that the 3T3-TNR9 cells display levels of tetradecanoyl phorbol acetate binding and of a calcium- and phospholipid-dependent protein kinase activity which are at least the equal of those seen in the parental 3T3 cells, implicating some postreceptor event in the nonmitogenic phenotype. In addition, we find that phosphorylation of the epidermal growth factor receptor and of 80-kDa and 22-kDa proteins, as well as the tyrosine phosphorylation of a 42-kDa protein, all proceed normally in the nonmitogenic variant, even though these phosphorylations must depend on the activation of different kinases. Thus, all these early phosphorylation reactions are intact in the 3T3-TNR9 cells. Although these phosphorylations may be necessary, they clearly are insufficient to trigger mitogenesis.

Granulocyte-macrophage colony-stimulating factor and tetradecanoyl phorbol acetate induce a distinct, restricted subset of primary-response TIS genes in both proliferating and terminally differentiated myeloid cells.

Induction of early-response genes (tetradecanoyl phorbol acetate [TPA]-induced sequences, or TIS genes; R.W. Lim, B.C. Varnum, and H.R. Herschman, Oncogene 1:263-270, 1987) by granulocyte-macrophage colony-stimulating factor (GM-CSF) and TPA was examined both in a factor-dependent murine cell line, 32D clone 3, and in mature human neutrophils. When GM-CSF-deprived 32D clone 3 cells were exposed to GM-CSF or to TPA, four TIS mRNAs (TIS7, TIS8, TIS10, and TIS11) were rapidly and transiently induced. However, neither GM-CSF nor TPA could induce accumulation of TIS1 mRNA in 32D clone 3 cells, even under superinducing conditions. Both GM-CSF and TPA also elicited rapid, transient expression of TIS8 and TIS11 mRNA in postmitotic human neutrophils. However, neither agent could induce accumulation of TIS1 mRNA in human neutrophils. TIS1 is a member of the nuclear receptor supergene family that codes for ligand-dependent transcription factors. Cell-type restriction of inducible transcription factors may contribute to developmental specification.

Functional and biochemical analysis of the C2 domains of synaptotagmin IV.

Synaptotagmins (Syts) are a family of vesicle proteins that have been implicated in both regulated neurosecretion and general membrane trafficking. Calcium-dependent interactions mediated through their C2 domains are proposed to contribute to the mechanism by which Syts trigger calcium-dependent neurotransmitter release. Syt IV is a novel member of the Syt family that is induced by cell depolarization and has a rapid rate of synthesis and a short half-life. Moreover, the C2A domain of Syt IV does not bind calcium. We have examined the biochemical and functional properties of the C2 domains of Syt IV. Consistent with its non-calcium binding properties, the C2A domain of Syt IV binds syntaxin isoforms in a calcium-independent manner. In neuroendocrine pheochromocytoma (PC12) cells, Syt IV colocalizes with Syt I in the tips of the neurites. Microinjection of the C2A domain reveals that calcium-independent interactions mediated through this domain of Syt IV inhibit calcium-mediated neurotransmitter release from PC12 cells. Conversely, the C2B domain of Syt IV contains calcium binding properties, which permit homo-oligomerization as well as hetero-oligomerization with Syt I. Our observation that different combinatorial interactions exist between Syt and syntaxin isoforms, coupled with the calcium stimulated hetero-oligomerization of Syt isoforms, suggests that the secretory machinery contains a vast repertoire of biochemical properties for sensing calcium and regulating neurotransmitter release accordingly.

Adrenergic, cholinergic, and inactive human neuroblastoma cell lines with the action-potential Na+ ionophore.

Cultured human neuroblastoma cell lines were assayed for biochemical characteristics of neuonal function. Cell lines studied included LA-N-1, LA-N-2, IMR-32, SK-N-SH, and SK-N-MC. Veratridine-dependent uptake of 22Na+ implied the presence of the action potential Na+ ionophore in LA-N-1, LA-N-2, IMR-32, and SK-N-SH. The time course of 22Na+ uptake and inhibition of uptake by tetrodotoxin supported this. SK-N-MC had no veratridine-dependent 22Na+ uptake. Tyrosine hydroxylase (EC 1.14.10.), glutamic acid decarboxylase (EC 4.1.1.15), and acetylcholine contents in neuroblastoma cells were compared to those in brain. LA-N-1 and IMR-32 contained 15 and 5 times as much tyrosine hydroxylase, respectively, whereas LA-N-2, SK-N-SH, and SK-N-MC contained only 0.5 to 5% of that in brain. Acetylcholine was present in -LA-N-2 in 15- to 20-fold greater quantities than in brain; other lines had only 10 to 50% of that in brain. None of the cell lines contained glutamic acid decarboxylase. Thus, continuously propogated human neuroblastoma cell lines may have the action potential Na+ ionophore and may be adrenergic (LA-N-1 and IMR-32), cholinergic (LA-N-2), or inactive (SK-N-SH and SK-N-MC). This is the first demonstration of the action potential Na+ ionophore and of acetylcholine production in human neuroblastoma cell lines.