Biomedical subjects

I Tamm

Publications and source records attributed to I Tamm.

At least 19 recordsLinked to original sources

PubMed1992

Molecular cloning and characterization of additional factors that bind to the interferon-alpha/beta response element B of the murine (2'-5')oligoadenylate synthetase ME-12 gene.

The function of interferon (IFN) response elements (IREs) may be classified into two classes according to the location of the IRE in the polymerase II transcription promoter: located upstream of the core promoter, or located within the core promoter. Thus, the IRE-binding factors (IREBFs) can serve as sequence-specific transcription factors that activate IFN-inducible gene transcription and they can participate in preinitiation complex formation. In the murine and human (2'-5')oligoadenylate synthetase genes the IRE is located within the core promoter region. For this reason, it is important to isolate cDNAs encoding protein factors that can bind to this IRE and determine their functional roles in the regulation of expression of the IFN-inducible (2'-5')oligoadenylate synthetase gene. We have isolated three groups of cDNAs that encode IREBFs. Here we report on clone 38 cDNA, which contains an open reading frame encoding a protein of 277 amino acids that is designated IREBF-2. The C terminus of IREBF-2 is extremely proline-rich and contains a high percentage of short-armed amino acids. IREBF-2 shares marked similarity with some gene products of the herpes group of viruses. These gene products are potent transcription activators. IREBF-2 is constitutively expressed in BALB/c 3T3 cells.

2',5'-Oligoadenylate Synthetase↗

PubMed1992

Dissociation between early loss of actin fibres and subsequent cell death in serum-deprived quiescent Balb/c-3T3 cells.

Serum withdrawal from either growing or quiescent Balb/c-3T3 murine fibroblasts causes a loss of F-actin fibres and focal adhesions within 30 min. Cells that are growing survive serum deprivation, whereas the great majority of density-arrested quiescent cells die during a period of up to 5 h from serum withdrawal. During this time an approximately constant fraction of the quiescent cell population dies per unit time. The population half-life is 60-70 min during this time. Addition of an appropriate cell growth factor or second messenger agonist at the time of serum withdrawal or within 2 h after serum withdrawal protects a similar fraction of viable cells. These findings suggest a model according to which withdrawal of serum (i.e. growth factors) initiates the death process in cells of the population with kinetics that approximate first-order kinetics. We postulate that appropriate growth factors or second messenger agonists block the initiating event that starts the cell death process.

3T3 Cells↗

PubMed1992

Identification of a new interferon-alpha/beta-inducible DNA-binding protein that interacts with the regulatory element A of 2',5'-oligoadenylate synthetase ME-12 gene.

A new interferon (IFN)-stimulated response factor (ISRF) has been identified in nuclear extracts of IFN-alpha/beta-treated murine BALB/c-3T3 fibroblasts by the mobility-shift electrophoresis assay. The factor, ISRF-2, displays murine 2',5'-oligoadenylate (2-5A) synthetase ME-12 gene 5' regulatory element A specificity and differs from the previously described IFN response element B-specific factor ISRF-1 in several aspects. ISRF-2 is restricted to the nucleus, whereas ISRF-1 exists in the cytoplasm and translocates into the nucleus upon treatment of cells with IFN-alpha/beta. The ionic strength requirement of ISRF-2 for maximal DNA-binding activity is lower than that of ISRF-1. The DNA-binding activity of ISRF-2, but not that of ISRF-1, is markedly suppressed by Mg2+. In common with ISRF-1, the phosphorylated form of ISRF-2 appears to be required for DNA-binding activity. A model is proposed for the mechanism whereby murine IFN-alpha/beta regulates 2-5A synthetase ME-12 gene expression.

2',5'-Oligoadenylate Synthetase↗

PubMed1991

Decreased adherence of interleukin 6-treated breast carcinoma cells can lead to separation from neighbors after mitosis.

Interleukin 6 (IL-6) has been shown to inhibit the proliferation, but increase the motility, of wild-type ZR-75-1 human ductal breast carcinoma cells, a line of cells that resemble closely the malignant cells cultured from the ascitic effusion. IL-6-treated cells lose their epithelial character, become stellate or fusiform in shape, and migrate away from neighbors. In the wild-type ZR-75-1 cells, IL-6 causes cell-cell separation in preformed colonies as well as postmitotically. We have now investigated the action of IL-6 in clone B ZR-75-1 cells, which are morphologically distinct from wild-type ZR-75-1 cells. In the more polygonal rather than cuboidal clone B cells, IL-6 did not cause early inhibition of DNA synthesis and it caused little cell-cell separation in preformed colonies. However, IL-6 treatment markedly prolonged the interval between mitosis and readherence of daughter cells to their neighbors and the substratum. Supernatants from IL-6-treated cultures contained detached viable cells in increased numbers. Intermitotic intervals were prolonged in IL-6-treated cultures. IL-6-treated dividing breast carcinoma cells are characterized by an increased probability of separation from neighbors and the substratum.

Breast Neoplasms↗

PubMed1991

Acidic and basic fibroblast growth factors are survival factors with distinctive activity in quiescent BALB/c 3T3 murine fibroblasts.

Platelet-derived growth factor (PDGF), epidermal growth factor, and insulin-like growth factor have previously been identified as survival factors with distinctive activities for the density-inhibited quiescent BALB/c 3T3 murine fibroblasts. Fibroblast growth factor (FGF), like PDGF, renders quiescent BALB/c 3T3 cells competent to respond to epidermal growth factor and insulin-like growth factor, which mediate cell-cycle traverse through G1 into S phase [Stiles, C. D., Pledger, W. J., VanWyk, J. J., Antoniades, H. N. & Scher, C. D. (1979) Proc. Natl. Acad. Sci. USA 76, 1279-1283]. We now show that FGF possess marked cell survival-enhancing activity distinctive from that of PDGF. Both acidic FGF (aFGF) and basic FGF (bFGF) markedly enhance short-term (3-hr) survival of quiescent cells. bFGF is the more active of the two factors and shows marked long-term (20-hr) survival-promoting activity alone, whereas aFGF requires heparin for long-term activity. Protection by bFGF or aFGF plus heparin is not associated with cell-cycle traverse into S phase. Both the short-term (3-hr) and long-term (20-hr) protective actions of aFGF and bFGF critically depend on protein synthesis, whereas those of PDGF do not. The accumulated evidence shows that several growth factors can contribute to maintenance of the integrity of quiescent murine fibroblasts and that their action can involve protein kinase A- and C-mediated processes as well as protein synthesis. Different growth factors display distinctive modes of action.

Alkaloids↗

PubMed1991

Molecular cloning and characterization of interferon alpha/beta response element binding factors of the murine (2'-5')oligoadenylate synthetase ME-12 gene.

Seven clones encoding interferon response element binding factors have been isolated from a mouse fibroblast lambda gt11 cDNA library by using a 32P end-labeled tandem trimer of the mouse (2'-5')oligoadenylate synthetase gene interferon response element as a probe. Clone 16 shares strong similarity (95%) at both DNA and amino acid level with YB-1, a human major histocompatibility complex class II Y-box DNA-binding protein, and with dbpB, a human epidermal growth factor receptor gene enhancer region binding protein. The product of the gene represented by clone 16 may represent a factor that regulates multiple genes by binding to a variety of 5' regulatory elements. Clone 25 is a 2407-base-pair-long cDNA and contains a putative 311-amino acid open reading frame corresponding to an estimated mass of 35.5 kDa. This putative protein, designated as interferon response element binding factor 1 (IREBF-1), contains an acidic domain, three heptad repeat leucine arrays, and a region that shares similarity with the yeast transcriptional factor GAL4 DNA-binding domain. Furthermore, the C terminus of IREBF-1 shows an unusual amphipathic property: within a 79-amino acid range, one side of the alpha-helical region contains a preponderance of hydrophobic amino acids and the other side contains hydrophilic amino acids. This type of structure provides a strong hydrophobic force for protein-protein interaction.

2',5'-Oligoadenylate Synthetase↗

PubMed1991

Effects of interferon-beta on the translocation rate and stationary time in human fibroblasts in culture.

The rate of translocation and the percent of the time that cells are stationary have been measured by computer-assisted time-lapse cinemicrography in over 1,000 freshly planted human foreskin fibroblasts (FS-4 cell strain) for periods of up to a week and the effects of interferon-beta (IFN-beta) on these parameters have been determined. Cells were planted at 2.5 X 10(3) cells/cm2 in Eagle's minimal essential medium (MEM) with 10% fetal bovine serum (FBS). Frames were taken every 2 or 4 minutes and data were collected on both cell location and cell division as a function of time. After planting FS-4 cells require approximately 48 hr to reach maximum motility both with respect to the translocation rate when moving and percent time cells are moving. Recombinant human INF-beta (800 mu/ml) caused a marked increase in the fraction of time cells were stationary and a decrease of lesser magnitude in the translocation rate, as quantitated during the period during which the stationary fraction for control cells was at a minimum. IFN-beta also decreased the rate of cell proliferation, without any evidence of degeneration or death of cells. Our results contribute new evidence that the fraction of time cells spend moving directionally is an important determinant of their locomotory behavior and that this determinant is responsive to modulation by cytokines.

Cell Division↗

PubMed1991

Expression and function of interleukin-6 in epithelial cells.

Epithelial cells both produce and are affected by interleukin-6 (IL-6). Experiments with an adenocarcinoma-derived cell line (HeLa) reveal that activation of the transfected human IL-6 promoter occurs largely through two partially overlapping second messenger (cAMP, phorbol ester)- and cytokine (IL-1, TNF, serum)-responsive enhancer elements (MRE 1, -173 to -151 and MRE II, -158 to -145). MRE I contains the typical GACGTCA cAMP and phorbol ester-responsive (CRE-TRE) motif, whereas MRE II defines a new CRE/TRE motif that contains an imperfect dyad repeat. The mechanism of dexamethasone-mediated repression of IL-6 gene expression in epithelial cells involves occlusion of the entire MRE enhancer region and of the core-promoter elements (TATA-box and RNA start site) by ligand-activated glucocorticoid receptor. Enhanced levels of IL-6 expression are observed in many solid tumors and in the hyperproliferative (and glucocorticoid-suppressible) lesions of psoriasis. In cell culture, IL-6 enhances, inhibits, or has no effect on the proliferation of epithelial cells depending upon the cell-type examined. IL-6 enhances proliferation of keratinocytes but inhibits that of breast carcinoma cell lines ZR-75-1 and T-47D. In these breast carcinoma cells, IL-6 elicits a major change in cell phenotype which is characterized by a fibroblastoid morphology, enhanced motility, increased cell-cell separation, and decreased adherens type junctions (desmosomes and focal adhesions). The new data identify IL-6 as a regulator of epithelial cell growth and of cell-cell association.

Animals↗

PubMed1991

Activation of signal transduction pathways protects quiescent Balb/c-3T3 fibroblasts against death due to serum deprivation.

Platelet-derived growth factor (PDGF), epidermal growth factor (EGF), insulin-like growth factor-1 (IGF-1), and insulin protect density-inhibited murine Balb/c-3T3 fibroblasts against death by distinctive mechanisms. Determination of the cell survival-enhancing activity of growth factors by cell enumeration and neutral red uptake measurement gives equivalent results. PDGF displays a steep dose-response relationship in the 1-5 ng/ml range. The other factors display shallow log-linear relationships in the following ranges: EGF: 0.2-5 ng/ml; IGF-1: 2-80 ng/ml; and insulin: 57-4,500 ng/ml. Agonists that lead to the activation of protein kinase A, including forskolin, 8-bromoadenosine 3':5'-cyclic monophosphate (Br-cAMP) and N6,2'-O-dibutyryladenosine 3':5'-cyclic monophosphate (db-cAMP), markedly increase both short-term (5-h) and long-term (20-h) survival of cells. 2-Isobutyl-1-methylxanthine (IBMX) markedly enhances short-term survival, but its effect decays with time. The protein kinase C agonist 12-O-tetradecanoyl phorbol-13-acetate (TPA) has a moderate protective effect at concentrations of 16-32 nM, and 64 nM TPA is highly effective. The synthetic diaclglycerols 1,2-dioctanoylglycerol (DiC8) and 1-oleoyl-2-acetylglycerol (OAG) and the calcium ionophore ionomycin show low activity. Supplementation of EGF with a protein kinase A or C agonist results in a varying additive increase in short-term (5-h) cell survival and supplementation of EGF + insulin or PDGF + EGF + insulin increases further the already high level of protection given by the growth factor combinations. Combining a protein kinase A and a protein kinase C agonist in the absence of growth factors gives an approximately additive increase in cell survival. Results obtained with kinase, RNA, and protein synthesis inhibitors suggest that: 1) activated protein kinase C catalyzes one or more phosphorylation events in quiescent Balb/c-3T3 cells that lead to gene expression with the protein product(s) mediating protection of quiescent cells against death, and 2) phosphorylation events catalyzed by protein kinase A largely serve to protect cells by a mechanism not requiring de novo RNA and protein biosynthesis.

2-Aminopurine↗

PubMed1991

Interleukin-6 enhances motility of breast carcinoma cells.

Interleukin-6 (IL-6) is the major systemic mediator of the early host response to infection and injury (the "acute phase response"). Furthermore, IL-6 is often detected in the peripheral circulation and in the local neoplastic tissue in cancer patients. IL-6 has distinctive effect on epithelial cells depending upon the cell type examined. IL-6 enhances proliferation of normal human keratinocytes without affecting cell morphology. In contrast, IL-6 inhibits the proliferation of ductal breast carcinoma cell lines T-47D, ZR-71-1 and MCF-7. In addition to, but independent of, the inhibition of cell proliferation, IL-6 induces a cellular phenotype in the typically epitheliod T-47D and ZR-75-1 cells, which is characterized by fibroblastoid morphology, increased cell-cell separation even within preformed colonies, decreased adherens type junction formation (desmosomes and focal adhesions), and enhanced motility. Time-lapse cinemicrography of T-47D and wild-type ZR-75-1 cells reveals increased local movement of IL-6-treated cells and also movement of these cells over considerable distances. The effects of IL-6 on breast cancer cell proliferation and motility are reversible by removal of IL-6 from the culture medium. Time-lapse cinemicrography reveals that in clone B ZR-75-1 cells, which are not sensitive to the DNA synthesis-inhibitory effect of IL-6 or to its cell-separating effect on preformed colonies, IL-6 can still block rapid readherence of post-mitotic cells to their neighbors and to the substratum leading to enhanced dispersal of cancer cells into the culture medium. In wild-type ZR-75-1 cells, 12-O-tetradecanoyl phorbol ester (TPA) exerts a cell-scattering effect on breast cancer cells without inhibiting cell proliferation. Combined treatment with IL-6 and TPA produces a cell-scattering effect that greatly exceeds in magnitude and speed the phenotypic change elicited by either reagent alone. Staurosporine blocks cell-scattering caused by TPA but not that caused by IL-6 suggesting that IL-6 and TPA elicit similar phenotypic changes in breast cancer cells via different pathways. Taken together, these findings identify a previously unrecognized property of IL-6, that of enhancing cell motility.

Breast Neoplasms↗

PubMed1991

Interleukin-6 and 12-O-tetradecanoyl phorbol-13-acetate act synergistically in inducing cell-cell separation and migration of human breast carcinoma cells.

Interleukin-6 (IL-6) causes an epithelial to fibroblastoid conversion and an increase in the motility of human ductal breast carcinoma cell lines ZR-75-1 and T-47D. Although IL-6 decreases DNA synthetic activity in these cell lines, the IL-6-induced alterations in cell shape and motility occur independently of inhibition of DNA synthesis per se. Whereas tumor necrosis factor alpha (TNF-alpha) inhibits DNA synthesis in T-47D cells, it does not cause an epithelial-fibroblastoid conversion or other major morphological changes and does not increase cell motility; TNF-alpha rapidly lyses a majority of ZR-75-1 cells. Furthermore, the DNA synthesis inhibitors 5-fluoro-2'-deoxyuridine (FUDR) and methotrexate (MTX) also do not cause effects mimicking the action of IL-6 on cell structure and motility. Transforming growth factors alpha and beta 1, acidic and basic fibroblast growth factors, epidermal growth factor, and insulin-like growth factor-1 (TGF-alpha, TGF-beta 1, aFGF, bFGF, EGF, and IGF-1) have little or no effect on breast cancer cell morphology, which serves to exclude the possibility that the IL-6-induced changes are a consequence of induction of these growth factors by IL-6. 12-O-tetradecanoyl phorbol-13-acetate (TPA) but not 8-bromoadenosine 3',5'-cyclic monophosphate (Br-cAMP) induces changes in the morphology and associative behavior of ZR-75-1 cells that are similar but not identical to those caused by IL-6. The TPA-induced alterations are not blocked by anti-IL-6 neutralizing antibodies; staurosporine inhibits the TPA-induced cell alterations but not those induced by IL-6. IL-6 and TPA used together have a phenotypic effect that greatly exceeds that of either agent alone and results in extensive cell scattering in less than 1 day. These findings are consistent with the hypothesis that IL-6 and TPA induce similar morphological changes and cell scattering via independent pathways.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1991

Effects of metals on the binding of protein factors to the mouse 2',5'-oligoadenylate synthetase ME-12 gene regulatory region.

We have previously demonstrated that using mouse 2',5'-oligoadenylate synthetase (2-5A synthetase) gene (ME-12) regulatory region AB as a probe, at least six complexes (C1-C6) can be detected by the mobility shift electrophoresis assay with extracts of murine BALB/c-3T3 cells. The formation of these complexes is greatly influenced by different buffer conditions and the presence of metals. C1 and C4 signals are stronger in Tris-HCl buffer than in phosphate buffer in the absence of added metals. Both Na+ and Mg2+ facilitate C1, C3, and C4 formation. Na+ also facilitates C3 and C4 formation with the interferon (IFN) response element B. Na+ has little effect on C2 formation with the regulatory region AB, whereas presence of Mg2+ at low concentrations gives a strong C2 signal. A new band, C7, was detected in the presence of 50 or 100 mM Na+. A relatively high Mg2+ concentration is required for C5 formation and Mg2+ interferes with C6 and C7 formation. Cu2+ and Zn2+ markedly inhibit formation of most complexes. However, a new band forms in 0.15 mM Cu2+ when 10 mM Mg2+ was also present. Co2+ enhances C2b and C3 formation and inhibits C4 formation. Mn2+ enhances C3, C1, and C2 formation to different degrees and inhibits C4 formation. At 0.15 and 0.25 mM, Mn2+ generates a new band. In competition experiments between Cu2+ and Mg2+, Cu2+ strongly inhibited Mg2+ effects on complex formation, except that 0.15 or 0.25 mM Cu2+ enhanced the C2a and C2b signals.

2',5'-Oligoadenylate Synthetase↗

PubMed1990