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.

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.

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.

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.

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.

Nucleotide sequence of a cDNA encoding TIS11, a message 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 TIS11, a gene induced in 3T3 cells by tetradecanoyl phorbol acetate.

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.

Megakaryocyte growth and development factor (MGDF) moderately enhances in-vitro platelet aggregation.

Megakaryocyte growth and development factor (MGDF) is a novel cytokine which promotes the development of immature megakaryocytes into platelets. We tested the hypothesis that MGDF would alter the sensitivity of platelets to aggregating agents as assessed by in-vitro platelet aggregometry. Platelet aggregation in the presence or absence of MGDF was tested with single doses of clinically relevant aggregating agents. A dose-dependent enhancement of the aggregation response to epinephrine was noted in MGDF treated platelets. When a range of concentrations of ADP were used to generate an aggregation dose-response curve, the addition of MGDF to platelet rich plasma shifted the dose response curve to the left. The effect of MGDF on platelet aggregation was partially prevented by the coincubation of platelets with a soluble form of the receptor for MGDF, the extracellular domain of c-mpl. In addition, we demonstrate that exogenous MGDF is able to induce tyrosine phosphorylation of platelet proteins with apparent molecular weights of 85 kDa and 130 kDa. From these data we conclude that exogenously added MGDF moderately increases the sensitivity of platelets to aggregating agents through a mechanism which appears to involve tyrosine phosphorylation of platelet proteins.

Synthesis, degradation, and subcellular localization of proteins encoded by the primary response genes TIS7/PC4 and TIS21/PC3.

Murine TIS7 and TIS21 cDNAs were cloned from phorbol ester-induced Swiss 3T3 cells. The cognate rat cDNAs, PC4 and PC3, were cloned from nerve growth factor (NGF)-treated PC12 pheochromocytoma cells. The TIS7/PC4 and TIS21/PC3 primary response genes are rapidly and transiently induced in response to serum, phorbol esters, and polypeptide growth factors in quiescent Swiss 3T3 cells and by NGF and other ligands in PC12 cells. In both 3T3 and PC12 cells the appearance of the TIS21/PC3 message precedes that of TIS7/PC4 message following ligand stimulation, suggesting that the TIS21/PC3 protein is likely to be synthesized more rapidly than the TIS7/PC4 protein. Using antisera prepared against recombinant TIS21 and TIS7 proteins, we find that the TIS21/PC3 protein is, indeed, synthesized more rapidly than the TIS7/PC4 protein following stimulation in both 3T3 and PC12 cells. In addition, "pulse-chase" experiments demonstrate that the TIS21/PC3 protein is degraded much more rapidly than the TIS7/PC4 protein. The sequences of the predicted PC3 and PC4 proteins have lead to the speculation that these two proteins may both be secreted from cells following stimulation. The PC4 protein is reported to have some sequence similarity to interferons. The TIS21/PC3 protein contains a presumptive leader sequence. Using our antisera to the recombinant proteins, however, we cannot detect secretion of radiolabelled TIS7/PC4 or TIS21/PC3 protein. Immunohistochemical and subcellular fractionation experiments suggest that the TIS7 protein is a membrane associated, non-nuclear intracellular protein. The TIS21 protein, in contrast, is a non-nuclear, soluble intracellular protein.

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.

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.

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.

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)

Axl receptor tyrosine kinase stimulated by the vitamin K-dependent protein encoded by growth-arrest-specific gene 6.

The Axl receptor tyrosine kinase was identified as a protein encoded by a transforming gene from primary human myeloid leukaemia cells by DNA-mediated transformation of NIH 3T3 cells. Axl is the founding member of a family of related receptors that includes Eyk, encoded by a chicken proto-oncogene originally described as a retroviral transforming gene, and c-Mer, encoded by a human proto-oncogene expressed in neoplastic B- and T-cell lines. The transforming activity of Axl demonstrates that the receptor can drive cellular proliferation. The function of Axl in non-transformed cells and tissues is unknown, but may involve the stimulation of cell proliferation in response to an appropriate signal, namely a ligand that activates the receptor. We report here the purification of an Axl stimulatory factor, and its identification as the product of growth-arrest-specific gene 6 (ref. 6). This is, to our knowledge, the first description of a ligand for the Axl family of receptors.

Human chondrocyte expression of growth-arrest-specific gene 6 and the tyrosine kinase receptor axl: potential role in autocrine signaling in cartilage.

OBJECTIVE: To determine if human articular chondrocytes express the axl tyrosine kinase receptor and its ligand Gas-6, a protein product of growth-arrest-specific gene 6, and to determine if Gas-6 and axl function in the regulation of chondrocyte growth and survival. METHODS: The presence of Gas-6 and axl was examined in situ in human articular cartilage by immunohistochemistry and in vitro in cell culture studies using primary human chondrocytes and immortalized human chondrocytes. The ability of recombinant Gas-6 to mediate adhesion of chondrocytes and to stimulate chondrocyte axl phosphorylation was determined. Studies of the role of Gas-6 and axl in cell proliferation and survival were also performed. RESULTS: Both Gas-6 and axl were detected in cartilage by immunohistochemical staining. Gas-6 and axl messenger RNA (mRNA) and protein were also detected in cultures of primary and immortalized human chondrocytes. Compared with cells cultured in medium containing 10% serum, Gas-6 mRNA levels were increased in immortalized chondrocytes cultured in serum-free medium, while axl expression decreased. Chondrocytes attached to Gas-6-coated plastic, and the attachment was blocked by a soluble Ig fusion protein containing the axl extracellular domain. Recombinant human Gas-6 and serum-free conditioned medium from primary and immortalized human chondrocyte cultures stimulated chondrocyte axl tyrosine phosphorylation. A mitogenic effect was noted both when immortalized chondrocytes were stimulated with recombinant Gas-6 or when they were made to overexpress axl by transfection. Addition of recombinant Gas-6 to serum-free medium resulted in increased survival of primary chondrocytes cultured at low density in agarose. CONCLUSION: These findings present evidence for an autocrine signaling pathway in cartilage involving Gas-6 and the axl tyrosine kinase adhesion receptor. Stimulation of axl by Gas-6 may play an important role in the control of chondrocyte growth and survival.

Novel neurotrophin-1/B cell-stimulating factor-3: a cytokine of the IL-6 family.

We have identified a cytokine of the IL-6 family and named it novel neurotrophin-1/B cell-stimulating factor-3 (NNT-1/BSF-3). NNT-1/BSF-3 cDNA was cloned from activated Jurkat human T cell lymphoma cells. Its sequence predicts a 225-aa protein with a 27-aa signal peptide, a molecular mass of 22 kDa in mature form, and the highest homology to cardiotrophin-1 and ciliary neurotrophic factor. The gene for NNT-1/BSF-3 is on chromosome 11q13. A murine equivalent to NNT-1/BSF-3 also was identified, which shows 96% homology to human NNT-1/BSF-3. NNT-1/BSF-3 mRNA is found mainly in lymph nodes and spleen. NNT-1/BSF-3 induces tyrosine phosphorylation of glycoprotein 130 (gp130), leukemia inhibitory factor receptor beta, and signal transducer and activator of transcription 3 in the SK-N-MC human neuroblastoma cells. NNT-1/BSF-3 shows activities typical of IL-6 family members. In vitro, it supports the survival of chicken embryo motor and sympathetic neurons. In mice, it induces serum amyloid A, potentiates the induction by IL-1 of corticosterone and IL-6, and causes body weight loss and B cell hyperplasia with serum IgG and IgM increase. NNT-1/BSF-3 is a gp130 activator with B-cell stimulating capability.