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R W Lim

Publications and source records attributed to R W Lim.

18 recordsLinked to original sources
PubMed1992

Involvement of tyrosine kinase and protein kinase C in platelet-activating-factor-induced c-fos gene expression in A-431 cells.

In A-431 cells, platelet-activating factor (PAF) induces the expression of c-fos and TIS-1 genes in both the absence and the presence of cycloheximide in a structurally specific and receptor-coupled manner. We have now investigated the molecular mechanisms of this response, particularly in relation to the role of protein kinases. Pretreatment of cells with genistein or methyl-2,5-dihydroxycinnamate (tyrosine kinase inhibitors) or staurosporine (a protein kinase C inhibitor) for 20 min abolished the c-fos expression induced by PAF. Interestingly, when genistein was added 90 s after addition of PAF, no inhibition was observed. Similarly, staurosporine did not inhibit c-fos expression when added 8 min after PAF addition to the cells. These inhibitions were dose-dependent (IC50 for staurosporine was 180 nM, and for genistein 50 microM). Simultaneous addition of PAF and phorbol 12-myristate 13-acetate (PMA) did not give a synergistic effect on c-fos expression. Pretreatment of cells with PMA had no effect on [3H]PAF binding, but abolished the PAF-induced gene expression. PAF-stimulated gene expression was desensitized if cells were pretreated with PAF. Interestingly, epidermal growth factor was able to stimulate c-fos expression in PAF-desensitized cells, and thus indicated involvement of distinct mechanisms for the two stimuli. Forskolin, an activator of adenylate cyclase, did not induce c-fos expression and had no effect on the PAF response. Exposure of cells to PAF for as little as 1 min, followed by its removal, was sufficient to activate the gene expression and demonstrated the rapidity and the exquisite nature of the signalling involved in this process. It is concluded that activation of PAF receptor (a proposed G-protein-coupled receptor) causes rapid production of signals which induce the expression of c-fos gene and that this is mediated via tyrosine kinase and protein kinase C.

Alkaloids↗
PubMed1991

Structure and expression of TIS21, a primary response gene induced by growth factors and tumor promoters.

The TIS21 gene is a primary response gene that is induced rapidly and transiently in 3T3 cells by the tumor promoter and mitogen tetradecanoyl phorbol acetate. The predicted open reading frame of the TIS21 cDNA encodes a protein of 158 amino acids with no obvious similarity to any known protein. Antiserum prepared to TIS21 recombinant protein produced in Escherichia coli precipitates a 17-kDa protein from Swiss 3T3 cells. The 2040-nucleotide 3'-untranslated region of the cDNA includes an unusual T18 sequence. The TIS21 gene has a single 1.4-kilobase intron which interrupts the open reading frame and is otherwise identical to the cDNA sequence. The 5'-flanking sequence of the TIS21 gene contains TATA and CAAT box-type sequences, three potential Sp1 sites, two putative cyclic AMP response elements, two potential AP1 binding elements, and an AP2 element. A possible Z-DNA structure of 29 AC repeats is present 660 nucleotides from the start of transcription. Expression from a luciferase reporter construct containing a 460-nucleotide fragment of the TIS21 promoter is induced by tetradecanoyl phorbol acetate, forskolin, epidermal growth factor, and serum, despite the absence of a consensus serum response element.

Amino Acid Sequence↗
PubMed1991

TIS10, a phorbol ester tumor promoter-inducible mRNA from Swiss 3T3 cells, encodes a novel prostaglandin synthase/cyclooxygenase homologue.

TIS10 is a primary response gene whose cDNA was cloned as a result of its rapid, superinducible expression in Swiss 3T3 cells in response to 12-O-Tetradecanoylphorbol-13-acetate. The 5'-untranslated region of the 3.9-kilobase TIS10 message contains only 124 nucleotides, whereas the 3'-untranslated region is almost 2 kilobases in length. Within this long 3' region, there are multiple repeats of the sequence ATTTA, a sequence often present in rapidly degraded mRNA species. Primer extension revealed that the TIS10 cDNA begins 16 base pairs downstream of the transcription start site for the TIS10 gene. The TIS10 cDNA encodes a predicted protein of 604 amino acids. A computer search identified striking similarities between the predicted TIS10 protein product and the murine, sheep, and human prostaglandin synthase/cyclooxygenase proteins. The TIS10 protein has many of the same conserved amino acids that are thought to be important for cyclooxygenase function. TIS10 mRNA is undetectable by Northern analysis in quiescent 3T3 cells. The TIS10 gene is rapidly and transiently induced by forskolin and serum, as well as by 12-O-tetradecanoylphorbol-13-acetate, in Swiss 3T3 cells. These agents elicit far more dramatic changes in TIS10 mRNA levels than in cyclooxygenase mRNA levels. The expression of the TIS10 gene appears to be highly cell type-restricted in cultured cell lines; of 12 cell lines tested under superinducing conditions, only the rodent embryonic Swiss 3T3 and Rat1 cell lines expressed TIS10 gene.

Amino Acid Sequence↗
PubMed1991

Platelet activating factor induces expression of early response genes c-fos and TIS-1 in human epidermoid carcinoma A-431 cells.

The effect of platelet activating factor (PAF) on the induction of early response genes was investigated in A-431 cells (human epidermal carcinoma cells). PAF induced a transient expression of c-fos and TIS-1 mRNA in a time- and dose-dependent manner. As low as 10(-10) M PAF caused detectable expression of these genes with a maximum observed at 10(-7) M. In the presence of cycloheximide, increases in the gene expression were noticeable at 20 min and peaked between 30-60 min. A lack of induction with lyso-PAF, an inactive PAF metabolite, confirmed the specificity of PAF towards this expression. The cells pretreated with CV-6209, a PAF receptor antagonist, did not show any induction of these genes by PAF. It is concluded that PAF causes induction of the early response genes c-fos and TIS-1 in a structurally specific and receptor dependent manner. This finding offers a new role for PAF at the nuclear level and may have important implications in the long term effects of PAF in pathophysiological conditions.

Cycloheximide↗
PubMed1989

TIS gene expression in cultured rat astrocytes: induction by mitogens and stellation agents.

The expression of a number of TIS genes (Lim et al.: Oncogene 1:263-270, 1987) was examined in secondary cultures of rat neocortical astrocytes treated with mitogens and stellation agents, to study the early nuclear events which accompany the induction of glial proliferation and/or differentiation. Tetradecanoyl phorbol acetate (TPA), epidermal growth factor, and fibroblast growth factor, three mitogens for astrocytes, stimulated marked, rapid, and transient increases in TIS gene mRNAS. TIS10, which is not expressed in rat PC12 pheochromocytoma cells, could be induced by these mitogens in rat astrocytes. Dibutyryl cyclic adenosine monophosphate and forskolin, which induce rapid stellation in astrocytes, and ganglioside GM1, a potent mitogen as well as an antagonist of the induction and maintenance of stellation, all induced TIS gene expression. Thus, a broad range of agents which elicit both proliferative and differentiation responses in astrocytes are capable of inducing a family of genes that may play a role in the early events of signal transduction.

Animals↗
PubMed1989

TIS gene expression in cultured rat astrocytes: multiple pathways of induction by mitogens.

Accumulation of TIS1 and TIS11 (Lim et al.: Oncogene 1:263-270, 1987) mRNAs in secondary cultures of rat neocortical astrocytes was much greater in response to tetradecanoyl phorbol acetate (TPA) than in response to either epidermal growth factor (EGF) or fibroblast growth factor (FGF). In contrast, EGF, FGF, and TPA were equally effective in inducing accumulation of TIS8 and TIS28/c-fos mRNAs. These data suggested that TPA and the polypeptide mitogens might induce TIS gene expression by distinct pathways. When maximally inducing concentrations of EGF and FGF were co-administered to astrocyte cultures, TIS mRNA accumulations were no greater than those observed for the individual growth factors, suggesting that EGF and FGF saturate a common, limiting step in their induction pathways. In contrast, when either EGF or FGF was presented to astrocytes in combination with maximally inducing levels of TPA, the resulting levels of accumulation of TIS mRNAs were at least as great as the sum of the levels induced by the individual mitogens. Stimulation of [3H]-thymidine incorporation demonstrated an identical pattern of interaction; EGF and FGF co-administration was no more effective than either polypeptide mitogen alone, but, when presented to astrocyte cultures along with maximally inducing concentrations of TPA, either EGF or FGF was able to increase incorporation of [3H]-thymidine. Superinduction of all the TIS genes occurred if cycloheximide (CHX) was present during TPA exposure. Once again, two distinct classes of responses of the various TIS genes occurred; superinduction of TIS1, TIS7, TIS11, and TIS28/c-fos mRNA accumulation ranged from 10- to 20-fold, while CHX superinduction of TIS8 and TIS10 was far more modest, ranging from 2- to 3-fold.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

The tumor promoters 12-O-tetradecanoylphorbol-13-acetate and okadaic acid differ in toxicity, mitogenic activity and induction of gene expression.

Okadaic acid (OA) and 12-O-tetradecanoylphorbol-13-acetate (TPA) are both potent tumor promoters in a mouse skin carcinogenesis experiment. OA was much more toxic than TPA for murine embryo cell lines such as Swiss 3T3 cells or C3H10T1/2 cells. TPA is a potent mitogen for 3T3 cells; in contrast OA was unable to stimulate DNA synthesis in these cells. TPA induces a family of primary response genes, the TPA induced sequence (TIS) genes, in a wide variety of cells. Although OA induced modest levels of TIS mRNA expression, the time course of the induction of TIS1 and TIS8 mRNA was delayed when compared to induction by TPA or peptide mitogens such as fibroblast growth factor (FGF). In addition TPA-mediated down-regulation of protein kinase C attenuated TIS gene induction by OA, but not by FGF.

Animals↗
PubMed1989

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.

Animals↗
PubMed1989

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.

Bone Marrow Cells↗
PubMed1989

Characterization of TIS7, a gene induced in Swiss 3T3 cells by the tumor promoter tetradecanoyl phorbol acetate.

We report here the nucleic acid sequence and deduced amino acid sequence of a cDNA for TIS7, a gene induced by mitogens in 3T3 cells and by nerve growth factor in PC12 pheochromocytoma cells. A fragment of the TIS7 gene has previously been cloned from murine fibroblast cells infected with Newcastle Disease Virus. The cDNA sequence suggests some similarities with murine interferons. TIS7 mRNA can also be transiently induced by double-stranded RNA. We suggest that the TIS7 protein may be an autocrine factor that attenuates or amplifies the initial ligand-induced signal.

Amino Acid Sequence↗
PubMed1988

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.

Animals↗
PubMed1987

Cloning of tetradecanoyl phorbol ester-induced 'primary response' sequences and their expression in density-arrested Swiss 3T3 cells and a TPA non-proliferative variant.

The tumor promoter tetradecanoyl phorbol acetate (TPA) is also a potent mitogen for murine 3T3 cells. We have previously described the isolation of variant Swiss 3T3 cell lines unable to proliferative in response to TPA. In this report we sought to identify genes that are stimulated by TPA as 'primary responses', i.e., without intervening protein synthesis. We constructed a cDNA library in lambda gt10, using RNA from quiescent 3T3 cells treated with TPA in the presence of cycloheximide (CHX). Of 50,000 recombinant phages, we identified 50 isolates that demonstrated preferential hybridization to cDNA probes generated from TPA-stimulated cells. One of the clones contains a fragment of the proto-oncogene c-fos. Twenty-nine of the remaining 49 clones fall into six cross-hybridization families. All the characterized clones detected mRNAs that are also inducible by epidermal growth factor, fibroblast growth factor or elevated serum. TPA induction of all the characterized messages is rapid and transient. All these mRNAs are superinduced by a combination of mitogens and CHX. Induction of these messages following TPA addition also occurs in subconfluent 3T3 cultures; expression of these genes is, therefore, not restricted to the G0/G1 transition. Expression of all six clones was also induced by TPA and other mitogens in 3T3-TNR9 cells, a TPA non-proliferative variant.

Animals↗
PubMed1987

Induction of transiently expressed genes in PC-12 pheochromocytoma cells.

Rat PC-12 pheochromocytoma cells, in response to nerve growth factor (NGF), stop proliferating and differentiate into cells resembling sympathetic neurons. This model of cell differentiation was used to investigate the expression of a previously isolated collection of mitogen-induced primary response sequences cloned from murine 3T3 cells; the TIS (tetradecanoyl phorbol acetate-induced sequences) genes (Lim et al., 1987). The TIS cDNAs were used to probe RNA isolated from PC-12 cells stimulated with NGF and other agents. Six of these messages were rapidly and transiently induced by NGF, tetradecanoyl phorbol acetate (TPA), or epidermal growth factor (EGF). Expression of these TIS genes generally resembled the NGF-stimulated induction of c-fos. In contrast, one TIS gene (TIS 10), induced by mitogens in 3T3 cells, was not induced by NGF, TPA, or EGF in PC-12 cells. Like c-fos, these TIS genes induced by NGF could also be superinduced by the combined administration of NGF and benzodiazepine. Elevated potassium ion, which leads to the induction of c-fos in PC-12 cells via activation of a voltage-dependent Ca2+ channel, also induces all TIS genes, with the notable exception of TIS 10. The induction of this family of genes may be involved in the general transduction of extracellular signals into biological responses.

Animals↗
PubMed1986

Epidermal growth factor-nonresponsive 3T3 variants do not contain epidermal growth factor receptor-related antigens or mRNA.

We have previously isolated three independent variants of Swiss 3T3 cells that are unable to generate a mitogenic response to epidermal growth factor (EGF). Each of the variants is unable to bind 125I-labeled EGF; each lacks a functional EGF receptor. We used an antiserum to murine EGF receptor to look for an EGF-receptor gene product in wild-type 3T3 cells and in the three EGF-nonresponsive variants. No cross-reactive material could be detected in any of the three variants, either in 125I-labeled cell extracts or in [35S]methionine metabolically labeled cells. 3T3 cells contained mRNA molecules homologous to a cDNA probe for the human EGF-receptor coding region. In contrast, no homologous RNA could be detected in any of the three variants. Analysis of genomic Southern blots of the DNA from 3T3 cells and the three EGF-nonresponsive variants indicated sequences from the EGF-receptor gene are present in the DNA of all four cell lines. These EGF-nonresponsive lines, which demonstrate proliferative responses to a variety of mitogens, will be ideal recipients for structure-function studies of the EGF receptor by transfection of the cloned gene.

Animals↗
PubMed1984

EGF responsiveness and receptor regulation in normal and differentiation-defective mouse myoblasts.

The interrelationship between cell proliferation and terminal myogenic differentiation has been analyzed by studying a differentiation-defective subclone (DD-1) of the permanent mouse myoblast line MM14. Parental MM14 myoblasts withdraw irreversibly from the cell cycle and initiate terminal differentiation when they are deprived of certain mitogens. In contrast, DD-1 cells become quiescent in a mitogen-depleted environment and less than 0.4% of the cells differentiate. When refed with mitogen-rich medium quiescent DD-1 cells resume proliferation. Expression of this differentiation-defective phenotype is apparently coupled to an alteration in mitogen sensitivity: MM14 myoblasts require horse serum plus either chick embryo extract or fibroblast growth factor (FGF) to sustain cell growth: DD-1 variants are responsive to FGF, but also proliferate in response to serum alone or to reduced serum plus epidermal growth factor (EGF). Interestingly, EGF also appears to retard DD-1 cell differentiation in a manner similar to the FGF repression of differentiation in normal myoblasts. Normal and differentiation-defective myoblasts which have been maintained under growth-promoting conditions exhibit similar EGF binding, internalization, and degradation. However, whereas the EGF binding capacity of MM14 myoblasts declines to less than 5% of its initial level within 24 hr of FGF removal, DD-1 variants exhibit an increase in EGF binding when switched to an FGF-depleted medium. The relationship of altered EGF receptor regulation to changes in mitogen sensitivity and differentiation capacity of the DD-1 variant is discussed, and implications for general in vivo processes governing cell proliferation and differentiation are considered.

Animals↗
PubMed1984

A rapid decrease in epidermal growth factor-binding capacity accompanies the terminal differentiation of mouse myoblasts in vitro.

Specific mitogens stimulate the proliferation and repress the differentiation of mouse myoblasts (MM14). When mitogens are depleted, MM14 cells cease proliferation, commit to terminal differentiation, and become refractory to growth stimulation. The behavior of mitogen receptors during the transition from a proliferative to a permanently postmitotic state was examined using the epidermal growth factor receptor (EGFR) as a model system. Whereas proliferating myoblasts bound substantial amounts of EGF, their binding capacity declined rapidly upon exposure to low-mitogen medium. The decline became irreversible when a cell differentiated. Within 24 h, less than 5% of the original EGF binding capacity remained. Since the ability to internalize and degrade bound EGF was unaffected, the change presumably reflected a decrease in EGFR availability. Several observations indicated that loss of EGFR following mitogen removal is related to differentiation rather than the result of starvation or cell-cycle arrest. First, the decline is correlated with the absence of a single mitogen (fibroblast growth factor) and is independent of serum concentrations. Second, myoblasts that are either cycling through G1 or arrested at G0, but prevented from differentiating, all bind large amounts of EGF. These findings suggest that specific reduction in mitogen receptors could be part of a mechanism whereby terminally differentiating cells become refractory to mitogenic stimulation.

Ammonium Chloride↗
PubMed1975

Application of latex microspheres in the isolation of plasma membranes. Affinity density perturbation of erythrocyte membranes.

Immunolatex spheres, originally developed as visual markers for scanning electron microscopy, were employed as membrane density perturbation reagents. Methacrylate spheres were bound to antibody molecules and used to label antigens on erythrocytes. Ghosts prepared from labeled cells were subjected to isopycnic centrifugation on continuous sucrose and dextran gradients. It was found that the labeled erythrocyte membranes had a substantially higher density than unlabeled membranes. The extent to which the membrane density was shifted on a given gradient depended on the number, size and density of the latex spheres and could be closely predicted by theory. These results suggest that the reagents and techniques described here have potential application for the isolation of plasma membranes from more complex cell types.

Animals↗