Early induction of ribonucleotide reductase gene expression by transforming growth factor beta 1 in malignant H-ras transformed cell lines.

Previous investigations have indicated that the suppression of proliferation by transforming growth factor (TGF) beta 1 is often lost upon cellular transformation, and that proliferation of some tumors is stimulated by TGF-beta. The present study provides the first observation of a link between TGF-beta 1 regulation of this process and alterations in the expression of ribonucleotide reductase, a highly controlled rate-limiting step in DNA synthesis. A series of radiation and T24-H-ras-transformed mouse 10T1/2 cell lines exhibiting increasing malignant potential was evaluated for TGF-beta 1 induced alterations in ribonucleotide reductase M1 and M2 gene expression. Early increases in M1 and/or M2 message and protein levels were observed only in malignant cell lines. The TGF-beta 1 induced changes in M1 and/or M2 gene expression occurred prior to any detectable changes in the rates of DNA synthesis, supporting the novel concept that ribonucleotide reductase gene expression can be elevated by TGF-beta 1 without altering the proportion of cells in S phase. T24-H-ras-transformed 10T1/2 cells were transfected with a plasmid containing the coding region of TGF-beta 1 under the control of a zinc-sensitive metallothionein promoter. When these cells were cultured in the presence of zinc, a large induction of TGF-beta 1 message was observed within 1 h. Both M1 and M2 genes were also induced, with increased mRNA levels appearing 2 h after zinc treatment, or 1 h after TGF-beta 1 message levels were clearly elevated. In total, the data suggests a mechanism of autocrine stimulation of malignant cells by TGF-beta 1, in which early alterations in the regulation of ribonucleotide reductase may play an important role.     

Alterations of ribonucleotide reductase activity following induction of the nitrite-generating pathway in adenocarcinoma cells

The murine adenocarcinoma cell line TA 3 synthesized nitrite from L-arginine upon stimulation with gamma-interferon (IFN-gamma) associated with tumor necrosis factor (TNF), and/or bacterial lipopolysaccharide (LPS), but not with IFN-gamma, TNF, or LPS added separately. Induction of the NO2(-)-generating activity caused an inhibition of DNA synthesis in TA 3 cells. This inhibition was prevented by the L-arginine analog N omega-nitro-L-arginine, which inhibited under the same conditions nitrite production by TA 3 cells. The TA 3 M2 subclone, selected for enhanced ribonucleotide reductase activity, was found to be less sensitive than the wild phenotype TA 3 WT to the cytostatic activity mediated by the NO2(-)-generating system. Cytosolic preparations from TA 3 M2 cells treated for 24 or 48 h with IFN-gamma, TNF, and LPS exhibited a reduced ribonucleotide reductase activity, compared to untreated control cells. No reduction in ribonucleotide reductase activity was observed when N omega-nitro-L-arginine was added to treated cells. Addition of L-arginine, NADPH, and tetrahydrobiopterin into cytosolic extracts from 24-h treated TA 3 M2 cells triggered the synthesis of metabolic products from the NO2(-)-generating pathway. This resulted in a dramatic inhibition of the residual ribonucleotide reductase activity present in the extracts. The inhibition was reversed by NG-monomethyl-L-arginine, another specific inhibitor of the NO2(-)-generating activity. No L-arginine-dependent inhibition of ribonucleotide reductase activity was observed using extracts from untreated cells that did not express NO2(-)-generating activity. These results demonstrate that, in an acellular preparation, molecules derived from the NO2(-)-generating pathway exert an inhibitory effect on the ribonucleotide reductase enzyme. This negative action might explain the inhibition of DNA synthesis induced in adenocarcinoma cells by the NO2(-)-generating pathway.     

Hypoxia-induced bFGF gene expression is mediated through the JNK signal transduction pathway

Although the synthesis of angiogenic factors in hypoxic regions of solid tumors is recognized as one of the critical steps in tumor growth and metastasis, the signal transduction pathway involved in hypoxic induction of basic fibroblast growth factor (bFGF) gene expression is still obscure. In the study described here, we investigated the intracellular responses to hypoxia and the mechanisms triggering the initiation of angiogenic activity in drug-resistant human breast carcinoma MCF-7/ADR cells. Northern blots showed an increase in the level of c-jun, c-fos, and bFGF mRNA during hypoxia. Gel mobility-shift analysis of nuclear extracts from hypoxia-exposed cells showed an increase in AP-1 binding activity. In addition, hypoxic treatment strongly activated c-Jun N-terminal kinase 1 (JNK1), leading to phosphorylation and activation of c-Jun. Expression of a dominant negative mutant of JNK1 suppressed hypoxia-induced JNK1 activation as well as bFGF gene expression. Taken together, hypoxia-induced bFGF gene expression is mediated through the stress-activated protein kinase (SAPK) signal transduction pathway.     

反效信号传导通路与氧化应激诱导的基因表达

细胞存活与凋亡受生长因子活化的ＥＲＫ与应激活化的ＪＮＫ－ｐ３８信号传导通路二者的动态平衡所控制,ＪＮＫ－ｐ３８通路的持续活化及ＥＲＫ通路的共济失活是细胞凋亡的关键所在。Ｈ２Ｏ２参与多种细胞类型的信号传导,并可诱导早期应答基因的表达。转录因子的激活与抗氧化酶合成有一定的因果关系,但中间环节尚待进一步阐明。     

The dose dependent effect of cyclic AMP on ribonucleotide reductase in mitogen stimulated mononuclear cells

Cyclic adenosine monophosphate arrests proliferating T lymphocytes in the G1 phase of the cell cycle. Here we demonstrate that exogenous and endogenous elevations in cyclic AMP concentration result in diminished mitogen stimulation, cell cycle arrest, and decreased ribonucleotide reductase messenger RNA concentrations in peripheral blood mononuclear cells. At lower concentrations (less than 1mM) of dibutyryl cyclic AMP that do not generate cell cycle arrest there is inhibition of ribonucleotide reductase activity without decreased messenger RNA concentration for the M2 subunit of ribonucleotide reductase. However, at higher concentrations of dibutyryl cyclic AMP there is G1 cell cycle arrest and reduced M2 specific messenger RNA concentration. Thus, cyclic AMP inhibition of lymphocyte activation may occur by different mechanisms that are dose dependent.     

Brassinosteroid signal transduction: clarifying the pathway from ligand perception to gene expression

Recent genetic screens for novel components of brassinosteroid signaling have revealed proteins with cell surface, cytoplasmic, and nuclear localization that function as either positive activators or negative regulators of the brassinosteroid response. Initial microarray experiments have expanded the number of known brassinosteroid-regulated genes, providing a useful resource for better understanding terminal events in signal transduction.     

Deoxyribonucleotide pools in mouse-fibroblast cell lines with altered ribonucleotide reductase.

Mutant cells lines of 3T6 mouse fibroblasts, resistant to thymidine and deoxyadenosine, have an altered allosteric regulation of the enzyme ribonucleotide reductase (Meuth, M. and Green, H., Cell, 3, 367, 1974). Compared to 3T6, these lines contain larger pools of deoxynucleoside triphosphates, in particular deoxycytidine triphosphate, but show a normal rate of DNA synthesis. Addition of thymidine or deoxyadenosine to 3T6 cells results in large accumulations of the corresponding triphosphates and a dramatic decrease in the dCTP pool, concomitant with inhibition of DNA synthesis. Addition of thymidine to the mutant cell lines also leads to an increase in the dTTP pool but does not result in a depletion of dCTP or inhibition of DNA synthesis. Addition of deoxyadenosine only leads to a small increase of the dATP pool. In general the change in the allosteric regulation of bibonucleotide reductase is reflected in the deoxynucleotide pools.     

Ethanolamine base exchange enzymatic activities in spontaneous transformed glial cell lines. Effect of dibutyryl cyclic AMP treatment

Cultured astrocytes derived from neonatal rats (normal cells) displayed maximal ethanolamine base exchange enzymatic activity (EBEE) when cultures reached confluency and cells almost ceased to divide. At this stage, ethanolamine phosphotransferase (EPT) and choline base exchange enzyme (CBEE) activities reached a plateau. In spontaneously transformed glial cells, no differential activity variation either between EPT and CBEE, or between EPT and EBEE was observed. The EBEE activity was mainly localized in the microsomal fraction and was completely absent from plasma membranes. Dibutyryl cyclic AMP (db-cAMP) treatment of the transformed cells reversed the pattern of these activities to that of normal cells. Moreover, treatment of the transformed cells with medium conditioned by normal astroblasts markedly increased EBEE activity. This study demonstrates that (i) variation of EBEE activity during cell growth differs in normal and in transformed cultured glial cells. (ii) EBEE activity may be modulated via both db-cAMP and normal cell conditioned medium. Our findings suggest a possible implication of EBEE in the maturation and contact inhibition of cell growth.     

Cyclic AMP levels in temperature sensitive SV40 transformed cell lines

The cAMP levels of the 3T3 cell line and its transformed derivatives were determined. The level was found to be lower in transformed cell lines than in parental 3T3 and to decrease at confluence in all cell lines tested. SV40 transformed 3T3 cell lines which are temperature sensitive in growth control were found to have lower cAMP levels than 3T3, but these levels were not temperature dependent. Retransformation of these cell lines by murine sarcoma virus did not markedly affect their cAMP levels.     

Alterations in the components of ribonucleotide reductase in hydroxyurea-resistant hamster cells

Two components of mammalian ribonucleotide reductase have been separated by blue dextran-Sepharose chromatography from a hydroxyurea-resistant cell line, NCR-30A2, and its parental wild type. Analysis of reductase activity in these cells and the enzyme components reveals that there are three alterations involving ribonucleotide reductase activity in NCR-30A2 cells. There is an elevation in the effector-binding (EB) component, an elevation in the non-heme-iron-containing (NHI) component, and an alteration in the NHI component that renders the enzyme less sensitive to inhibition by hydroxyurea. These findings easily account for the resistance of NCR-30A2 cells to the antitumor agent hydroxyurea, and to other drugs with a similar mode of action.     

Identification of pathway deregulation - gene expression based analysis of consistent signal transduction

Signaling pathways belong to a complex system of communication that governs cellular processes. They represent signal transduction from an extracellular stimulus via a receptor to intracellular mediators, as well as intracellular interactions. Perturbations in signaling cascade often lead to detrimental changes in cell function and cause many diseases, including cancer. Identification of deregulated pathways may advance the understanding of complex diseases and lead to improvement of therapeutic strategies. We propose Analysis of Consistent Signal Transduction (ACST), a novel method for analysis of signaling pathways. Our method incorporates information regarding pathway topology, as well as data on the position of every gene in each pathway. To preserve gene-gene interactions we use a subject-sampling permutation model to assess the significance of pathway perturbations. We applied our approach to nine independent datasets of global gene expression profiling. The results of ACST, as well as three other methods used to analyze signaling pathways, are presented in the context of biological significance and repeatability among similar, yet independent, datasets. We demonstrate the usefulness of using information of pathway structure as well as genes' functions in the analysis of signaling pathways. We also show that ACST leads to biologically meaningful results and high repeatability.     

Role of the JAK/STAT signal transduction pathway in the regulation of gene expression in CNS

Over the last 20 years the JAK/STAT signal transduction pathway has been extensively studied. An enormous amount of data on different cell signal transduction pathways is now available. The JAK/STAT signal transduction pathway is one of the intracellular signaling pathways activated by cytokines and growth factors that was first studied in the hematopoietic system, but recent data demonstrate that this signal transduction is also greatly utilized by other systems. The JAK/STAT pathway is a signaling cascade that links the activation of specific cell membrane receptors to nuclear gene expression. This review is focused on the role of JAK/STAT signal transduction pathway activation in the central nervous system (CNS).     

Stable luciferase reporter cell lines for signal transduction pathway readout using GAL4 fusion transactivators

While GAL4 fusion activators have been widely used for dissecting signal transduction pathways in transient assays, there has been surprisingly little reported on utilizing cell lines with stably integrated fusion activators. To avoid problems with the efficiency and reproducibility inherent to transient transfection, we describe here the generation and characterization of HeLa reporter cell lines, which contain a stably integrated luciferase gene responsive to stably integrated and constitutively expressed GAL4-CREB or GAL4-Elk1 fusion activators. These cell lines exhibited extremely low basal luciferase expression but robust response to various extracellular stimuli or the expression of signaling molecules that resulted in elevated MAP kinase or PKA activities. This integrated two-component reporter system allows one to focus specifically on particular signaling pathway endpoints and the altered transactivation activity of either Elk1 or CREB. With the procedures described here, many novel cell-based assays can be developed by generating new reporter cell lines with medically important but difficult-to-transfect cell types, and by using different reporter genes or different fusion transactivator genes.     

Involvement of cyclic AMP cell surface receptors and G-proteins in signal transduction during slug migration of Dictyostelium discoideum.

The presence of G-proteins, interacting with cAMP surface receptors, was investigated in vegetative cells, aggregation-competent cells, and migrating slugs of Dictyostelium discoideum. Our results indicate that G-proteins are present in all stages. In vegetative cells there is a limited number of cAMP receptors but no effect of GTP tau S on cAMP binding could be detected; in addition, no effect of cAMP on GTP tau S binding or GTPase activity was observed. In both aggregation-competent cells and slugs GTP tau S inhibits cAMP binding, while cAMP stimulates GTP tau S binding and high-affinity GTPase. Since the presence of G-proteins coupled to cAMP receptors could be demonstrated in slugs, the involvement of the effector enzymes adenylate cyclase and phospholipase C was investigated. The results show that adenylate cyclase activity is stimulated by GTP tau S in both stages and that in cells from migrating slugs the Ins(1,4,5)P3 production is increased upon stimulation with cAMP. The possible involvement of G-proteins in signal transduction during the slug stage of D. discoideum is discussed.