Biomedical subjects

H S Boswell

Publications and source records attributed to H S Boswell.

At least 19 recordsLinked to original sources

PubMed2002

Role for c-jun N-terminal kinase in treatment-refractory acute myeloid leukemia (AML): signaling to multidrug-efflux and hyperproliferation.

A relationship was proved between constitutive activity of leukemic cell c-jun-N-terminal kinase (JNK) and treatment failure in AML. Specifically, early treatment failure was predicted by the presence of constitutive JNK activity. The mechanistic origins of this association was sought. A multidrug resistant leukemic cell line, HL-60/ADR, characterized by hyperexpression of c-jun and JNK activity, was transfected with a mutant c-jun vector, whose substrate N-terminal c-jun serines were mutated. Down-regulated expression occurred of c-jun/AP-1-dependent genes, catalase and glutathione-S-transferase (GST) pi, which participate in cellular homeostasis to oxidative stress and xenobiotic exposure. MRP-efflux was abrogated in HL-60/ADR cells with dominant-negative c-jun, perhaps because MRP1 protein expression was also lost. Heightened sensitivity to daunorubicin resulted in cells subjected to this change. Biochemical analysis in 67 primary adult AML samples established a statistical correlation between cellular expression of c-jun and JNK activity, JNK activity with hyperleukocytosis at presentation of disease, and with exuberant MRP efflux. These findings reflect the survival role for c-jun/AP-1 and its regulatory kinase previously demonstrated for yeast in homeostatic response to oxidative stress and in operation of ATP-binding cassette efflux pumps, and may support evolutionary conservation of such function. Thus, JNK and c-jun may be salient drug targets in multidrug resistant AML.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2001

Transformation of interleukin-3-dependent cells without participation of Stat5/bcl-xL: cooperation of akt with raf/erk leads to p65 nuclear factor kappaB-mediated antiapoptosis involving c-IAP2.

Tyrosine kinase oncogenes such as p210BCR-ABL activate multiple signal pathways. As a result, it is difficult to infer the functional relevance of a pathway acting alone or in cooperation with another. One or 2 second-tier kinases represented in the p21ras and phosphatidylinositol-3-kinase (PI-3-kinase) pathways (activated RafCAAX and gag-akt, respectively) were expressed in parental H7 interleukin-3 (IL-3)-dependent myeloid cells. IL-3-dependent cells served, independently, as recipients of p210BCR-ABL, which activated p21ras and PI-3-kinase pathways, including raf/erk and akt, respectively, en route to transformation. By contrast, neither RafCAAX nor gag-akt when expressed in parental cells in isolation produced factor-independent cells. On the other hand, H7 cells expressing both RafCAAX and gag-akt (H7gag-akt/RafCAAX) were transformed. Such transformation in H7gag-akt/RafCAAX was accomplished in the absence of active versions of Shc or cbl, and there was no evidence of Stat activity and only modest amounts of bcl-xL, a Stat5 transcriptional target protein, all of which characterized the cells transformed by BCR-ABL. However, H7gag-akt/RafCAAX cells and H7BCR-ABL cells cultured in the absence of IL-3 shared strikingly increased p65 nuclear factor kappaB (NFkappaB) activity. Treatment of cells with a specific NFkappaB inhibitor, parthenolide, led to loss of NFkappaB activity and down-regulation of antiapoptotic c-IAP2. In cells with only gag-akt/RafCAAX, this was sufficient to allow polyADP ribosyltransferase (PARP)-degradative apoptosis, but in cells with p210BCR-ABL, apoptosis was blocked, possibly by a Stat5/bcl-xL-dependent mechanism. Therefore, one hematopoietic antiapoptotic program, among others, available to certain tyrosine kinase oncogenes involves a cooperative response between raf/erk and akt, unambiguous components of p21ras and PI-3-kinase pathways, to induce p65 NFkappaB and c-IAP2.

Animals↗

PubMed1999

Induction of apoptosis by arachidonic acid in chronic myeloid leukemia cells.

The hallmark of chronic myeloid leukemia (CML) is the presence of the bcr-abl oncogene, which is associated with transforming ability and an intrinsic resistance to induction of apoptosis by genotoxic agents. Arachidonic acid (AA), a biologically active fatty acid, plays a crucial role as a mediator of signaling pathways involved in cell proliferation and survival. In this study, we investigated the potential role of AA as a proapoptotic agent in CML. Pretreatment of human CML isolated progenitor cells with AA (100 microM for 18 h) induced 71-75% inhibition of in vitro colony formation of granulocyte-macrophage colony-forming units, multilineage colony-forming units, and erythroid burst-forming units. This inhibition was significantly greater than the effect on normal progenitor cells (19-39% growth inhibition of erythroid burst-forming units, multilineage colony-forming units, and granulocyte-macrophage colony-forming units). AA also inhibited growth of the bcr-abl-transformed cell line H7.bcr-abl A54. In contrast, a minimal effect of AA on inhibition of cell growth was observed in the parental nontransformed NSF/N1.H7 cell line. The antiproliferative effect of AA was associated with apoptosis. Gamma-linolenic acid, a precursor of AA, also inhibited cell growth, whereas other unsaturated and saturated fatty acids had no effect. Pharmacological inhibition of cyclooxygenase, lipooxygenase, and cytochrome P450 monooxygenase enzymes prior to exposure to AA did not rescue cells from the inhibitory effect of AA. Moreover, 5,8,11,14-eicosatetraynoic acid, a nonmetabolizable arachidonate analogue, also inhibited cell growth, suggesting that the effect of AA did not require further metabolism. Treatment with antioxidants prior to stimulation with AA was also ineffective in preventing its antiproliferative effect. Thus, AA inhibited proliferation of CML cells by inducing apoptotic cell death. The signaling mechanisms of AA-induced inhibition of cell growth appeared to be independent of its conversion into eicosanoids or free radical generation.

5,8,11,14-Eicosatetraynoic Acid↗

PubMed1998

Regulation of the c-jun gene in p210 BCR-ABL transformed cells corresponds with activity of JNK, the c-jun N-terminal kinase.

Activity of the c-jun N-terminal kinase (JNK) has been shown in hematopoietic cells transformed by p210 BCR-ABL. However, analysis has not been reported for hematopoietic cells on the consequences of this activity for c-jun promoter regulation within its distinctive proximal 8-base consensus CRE-like element, an element linked to JNK-mediated increase in c-jun transcription. In the present study, regulation of the proximal c-jun promoter was studied in murine myeloid cells transformed by p210 BCR-ABL. Promoter regulation in p210 BCR-ABL transformed cells was compared with regulation of the promoter in nontransformed interleukin-3 (IL-3)-dependent parental cells. The composition of nuclear AP-1 proteins contained within cells with p210 BCR-ABL, and their binding to the c-jun promoter proximal CRE-like element, was compared with the composition and binding of AP-1 proteins in IL-3-treated parental cells without p210 BCR-ABL. The present analysis found fivefold increased c-jun transcription occurring in p210 BCR-ABL transformed murine myeloid cells possessing a corresponding magnitude of increased kinase activity of JNK, compared with IL-3-stimulated parental cells. Augmented JNK activity was accompanied by increased nuclear abundance of c-jun and c-fos proteins that bound specifically to the proximal c-jun promoter CRE element. Also, representative human leukemic cell lines expressing p210 BCR-ABL and possessing abundant kinase activity of JNK, when compared with parental cells that were deficient in JNK activity, had increased c-jun and c-fos proteins. Finally, to show the relevance of these observations in model systems, we studied blast cells from patients with Philadelphia chromosome-positive acute leukemic transformation, and observed comparable activities of JNK catalysis and c-jun/AP-1 protein relative to the cell lines that possessed p210 BCR-ABL and JNK activity. These studies provide a basis for investigating the set of downstream genes which augmented c-jun/AP-1 activity enlists in the process of transformation by p210 BCR-ABL.

Animals↗

PubMed1997

Cyclic AMP negatively controls c-myc transcription and G1 cell cycle progression in p210 BCR-ABL transformed cells: inhibitory activity exerted through cyclin D1 and cdk4.

Raised intracellular cyclic AMP (cAMP) has been demonstrated to exert an antiproliferative effect in myeloid cells. How the antiproliferative activity of cAMP is exerted in p210 BCR-ABL transformed myeloid cells was the subject of this investigation. It was hypothesized that cyclin dependent kinase 4, cdk4, might be a critical target enzyme to affect the related events of c-myc transcription and progression through G1 phase of the cell cycle within cells transformed by p210 BCR-ABL, and further, that cdk4 might be downregulated by cAMP to inhibit proliferation. In order to investigate the regulatory role of cdk4, synchronized cells were studied. In p210 BCR-ABL transformed cells transiting early G1 phase, treatment with a cAMP analogue led to inhibition of cyclin D1 synthesis, and marked reduction of cdk4 kinase activity. Within cells in which cdk4 was inhibited by cAMP, there was augmented interaction of E2F1 with the retinoblastoma protein, pRb in a nuclear matrix-associated cell fraction. As a result of E2F1 sequestration, raised intracellular cAMP was found to inhibit c-myc transcription in p210 BCR-ABL transformed myeloid cells synchronously transiting the early G1 phase of the cell cycle. A target of this transcriptional suppression exerted by cAMP was the E2F site of the c-myc P2 promoter. On the other hand, cyclin D1 content was not reduced by cAMP in these cells when it was applied at a later cell cycle stage at the interface between G1 and S. Corresponding to lack of cyclin D1 inhibition in these later G1-to-S phase cells, cdk4 activity was only modestly suppressed, and c-myc mRNA expression was also inhibited to a lesser degree. These studies show that Rb interaction with E2F1 is regulated by cdk4 and cyclin D1 within p210 BCR-ABL transformed leukemia cells in early G1 phase of the cell cycle. In this context, both cyclin D1 and cdk4 are subject to the level of intracellular cAMP. This interaction between Rb and E2F1, which is subject to the level of cAMP, is critical to transcriptional control of c-myc. Further, pRb regulation of E2F activity affects cellular potential for G1-S phase transition in p210 BCR-ABL transformed myeloid cells, in part, via its effect on c-myc transcription.

Animals↗

PubMed1996

Steel factor induces serine phosphorylation of Stat3 in human growth factor-dependent myeloid cell lines.

Steel factor (SLF) acts synergistically with various hematopoietic growth factors that use the Jak-Stat pathways in vivo and in vitro, although the contribution by SLF to this pathway is unknown. We show here that SLF induces time- and dose-dependent phosphorylation of Stat3 in the human growth factor-dependent cell lines MO7e and TF-1. This phosphorylation occurs exclusively on serine residues. Simultaneous stimulation with SLF plus other cytokines that induce tyrosine phosphorylation of Stat3, such as interleukin-9 (IL-9) in MO7e cells or IL-6 in TF-1 cells, resulted in tyrosine phosphorylation and enhanced serine phosphorylation of Stat3. Serine phosphorylation alone did not promote nuclear translocation or DNA binding activity to the sis-inducible element of Stat3. However, costimulation with SLF plus IL-9 in MO7e cells resulted in the nuclear translocation of serine-hyperphosphorylated Stat3. Serine phosphorylation of Stat3 was also observed by the stimulation of cells with granulocyte-macrophage colony-stimulating factor and IL-3, which do not induce tyrosine phosphorylation of Stat3. These results suggest that SLF might modulate the Jak-Stat3 pathway by serine phosphorylation and that the Jak-Stat pathway may be differentially regulated by the combinational stimulation of two or more cytokines.

Base Sequence↗

PubMed1995

Arachidonic acid induces c-jun gene expression in stromal cells stimulated by interleukin-1 and tumor necrosis factor-alpha: evidence for a tyrosine-kinase-dependent process.

We have previously shown that granulocyte-macrophage colony-stimulating factor (GM-CSF) gene expression induced by interleukin-1 (IL-1) and tumor necrosis factor-alpha (TNF-alpha) in the murine stromal cell line +/+.1-LDA 11 involves activation of phospholipase A2 (PLA2). Furthermore, induction of GM-CSF gene expression due to release of arachidonic acid as a result of PLA2 activation was mediated by the transcriptional factor c-jun. In the present study, we have investigated the potential mechanism involved in the induction of c-jun gene expression by arachidonic acid. Arachidonic acid induced transcription of c-jun mRNA. Downregulation of protein kinase C (PKC) by chronic exposure of stromal cells to the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA; 400 nmol/L) did not effect c-jun expression induced by arachidonate. Moreover, pretreatment of cells with the PKC inhibitor, calphostin C (1 mumol/L), caused a marked decrease of c-jun expression induced by TPA, but had no influence on c-jun expression induced by arachidonate. To explore the hypothesis that a tyrosine kinase signalling pathway, independent of PKC activation, was involved in arachidonate-induced c-jun expression, stromal cells were pretreated with the protein tyrosine kinase inhibitor, genistein, before challenge with arachidonic acid. Arachidonate 50 mumol/L)-induced c-jun expression was inhibited, in a dose- and time-dependent manner, by genistein. Genistein similarly inhibited c-jun expression in stromal cells exposed to IL-1 (500 U/mL) plus TNF-alpha (500 U/mL). The potential role of a tyrosine kinase pathway in arachidonate-mediated c-jun expression was further investigated by assaying the tyrosine kinase activity of cells challenged with arachidonic acid, IL-1, and TNF-alpha. Exposure of stromal cells to arachidonic acid induced a 2.1-fold increase in intracellular tyrosine kinase activity determined by phosphorylation of the synthetic peptide, raytide, in the presence of [gamma-32P]-ATP. Similarly, IL-1 and TNF-alpha induced 1.7- and 2.4-fold increases in tyrosine protein kinase activity, respectively. The effect of arachidonic acid on tyrosine kinase activity was inhibited by genistein and was enhanced by sodium vanadate. The increase of protein tyrosine kinase activity detected in arachidonate-stimulated cells was associated, in a dose- and time-dependent fashion, with tyrosine phosphorylation of 240-, 40-, and 29-kD substrates. These results are consistent with the hypothesis that a tyrosine phosphorylation process is triggered by arachidonate as an early event in the signalling pathway that leads to increased expression of c-jun.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1995

Signal transduction during myeloid cell differentiation.

The intracellular signaling mechanisms that dictate myeloid differentiation and proliferation are discussed. Independent hematopoietic signaling pathways including p21ras pathway, c-myc pathway, and Jak-STAT pathway are defined. Emphasis is given to the process of information integration at the nucleus, by which developmental programs may be converted from binary decisions into the complex response patterns explaining hematopoietic diversity. Coupling between signaling and transcription is emphasized.

Animals↗

PubMed1995

Tyrosine phosphorylation and activation of focal adhesion kinase (p125FAK) by BCR-ABL oncoprotein.

Focal adhesion kinase (p125FAK; FAK) is a protein tyrosine kinase that is tyrosine-phosphorylated in response to v-src-mediated transformation, cell adhesion, and stimulation with neuropeptides. To elucidate a possible functional relationship between FAK and BCR-ABL oncoprotein detected in Philadelphia chromosome-positive (Ph+) leukemias, we investigated the tyrosine phosphorylation state of FAK in a murine growth factor-dependent cell line and in its stable human bcr-abl cDNA transfectant. In interleukin-3 (IL-3)-dependent NFS/N1.H7 cells, tyrosine phosphorylation of FAK was not detected after stimulation with either IL-3 or Steel factor (SLF), both of which involve Ras-mediated signaling pathways. However, stable gene transfection with p210bcr-abl cDNA into H7 cells made these cells growth factor-independent for proliferation and resulted in constitutive tyrosine phosphorylation and kinase activation of FAK. Constitutive phosphorylation and activation of FAK was also observed in all Ph+ leukemia cell lines examined--that is, K562, TS9;22, and YS9;22, which express p210BCR-ABL, and NALM-21 and OM9;22, which express p185BCR-ABL. Ph-negative (Ph-) cell lines, such as MO7e and JM, did not show any detectable tyrosine phosphorylation of FAK. FAK phosphorylation in BCR-ABL-expressing cells was inhibited in a dose-dependent manner by cytochalasin D, a reagent that disrupts the intracellular network of actin filaments. However, no suppression of kinase activity or protein expression of BCR-ABL was observed after treatment with cytochalasin D. A physical association between BCR-ABL and FAK was not apparent. These data suggest that BCR-ABL may be involved in the activation of FAK. Moreover, FAK may be distinct from components in Ras-mediated signaling cascades that are activated by stimulation of myeloid cells with various cytokines.

Animals↗

PubMed1995

Adeno-associated virus 2-mediated gene transfer and functional expression of the human granulocyte-macrophage colony-stimulating factor.

It is becoming increasingly clear that the adeno-associated virus 2 (AAV)-based vector system may prove to be useful for high-efficiency gene transfer in human cells as well as for potential gene therapy in humans. A recombinant AAV vector containing the gene for a human hematopoietic growth factor, granulocyte-macrophage colony-stimulating factor (GM-CSF), was constructed and used to infect COS-1 cells, a monkey kidney cell line. COS-1 cells infected with the recombinant virus, but not mock-infected cells, expressed high levels of the human GM-CSF gene transcripts. Furthermore, in co-cultivation experiments with the recombinant virus-infected cells, but not in those with mock-infected cells, active proliferation of a GM-CSF-dependent human megakaryocytic leukemia cell line, M07e, could be obtained in the absence of exogenously added GM-CSF. The recombinant GM-CSF protein released into the supernatant was biologically active in progenitor cell assays carried out with primary human hematopoietic cells, and this activity was specifically abrogated by treatment of the supernatant with anti-GM-CSF antibodies. This recombinant virus may be potentially useful in the management and gene therapy of a variety of malignant disorders in the human hematopoietic system.

Animals↗

PubMed1995

Role for E2F1 in p210 BCR-ABL downstream regulation of c-myc transcription initiation. Studies in murine myeloid cells.

Experiments were performed to elucidate the mechanism through which p210 BCR-ABL, by its downstream signals, regulates c-myc messenger RNA expression in hematopoietic cells. We studied a model system in which stable expression of p210 BCR-ABL in interleukin-3 (IL-3) dependent murine myeloid cell lines led to growth factor independent transformation. Active c-myc transcription was observed in p210 BCR-ABL transformed cells by nuclear run-on assay, and in heterologous reporter assays performed with the 5' regulatory region of murine c-myc linked to firefly luciferase. Transcription initiation occurred primarily from the P2 promoter in p210 BCR-ABL transformed cells. Cis and trans elements responsible for transcription initiation from the c-myc P2 promoter were studied. Expression of E2F1 protein in p210 BCR-ABL transformed cells accounted, in part, for binding to the E2F site of the P2 c-myc promoter. The functional importance of E2F1 expression in p210 BCR-ABL transformed cells toward c-myc transcription was established in reporter assays performed with the P2 c-myc promoter containing either wild-type or mutant E2F sites. Mutation of the E2F motif of P2 5' c-myc reduced activity of the promoter by 50%. By gel mobility shift, E2F1 was found in P2 c-myc band shift complexes along with the cyclin-dependent kinase 2. Therefore, coupling of E2F to components of the retinoblastoma-cyclin pathway defines a route from p210 BCR-ABL to c-myc transcription, which is required for p210 BCR-ABL transformation.

Animals↗

PubMed1994

Coupling between p210bcr-abl and Shc and Grb2 adaptor proteins in hematopoietic cells permits growth factor receptor-independent link to ras activation pathway.

Enforced expression of p210bcr-abl transforms interleukin 3 (IL-3)-dependent hematopoietic cell lines to growth factor-independent proliferation. It has been demonstrated that nonreceptor tyrosine kinase oncogenes may couple to the p21ras pathway to exert their transforming effect. In particular, p210bcr-abl was recently found to effect p21ras activation in hematopoietic cells. In this context, experiments were performed to evaluate a protein signaling pathway by which p210bcr-abl might regulate p21ras. It was asked whether Shc p46/p52, a protein containing a src-homology region 2 (SH2) domain, and known to function upstream from p21ras, might form specific complexes with p210bcr-abl and thus, possibly alter p21ras activity by coupling to the guanine nucleotide exchange factor (Sos/CDC25) through the Grb2 protein-Sos complex. This latter complex has been previously demonstrated to occur ubiquitously. We found that p210bcr-abl formed a specific complex with Shc and with Grb2 in three different murine cell lines transfected with a p210bcr-abl expression vector. There appeared to be a higher order complex containing Shc, Grb2, and bcr-abl proteins. In contrast to p210bcr-abl transformed cells, in which there was constitutive tight association between Grb2 and Shc, binding between Grb2 and Shc was Steel factor (SLF)-dependent in a SLF-responsive, nontransformed parental cell line. The SLF-dependent association between Grb2 and Shc in nontransformed cells involved formation of a complex of Grb2 with c-kit receptor after SLF treatment. Thus, p210bcr-abl appears to function in a hematopoietic p21ras activation pathway to allow growth factor-independent coupling between Grb2, which exists in a complex with the guanine nucleotide exchange factor (Sos), and p21ras. Shc may not be required for Grb2-c-kit interaction, because it fails to bind strongly to c-kit.

Adaptor Proteins, Signal Transducing↗

PubMed1994

p210 bcr-abl confers overexpression of inosine monophosphate dehydrogenase: an intrinsic pathway to drug resistance mediated by oncogene.

The p210 bcr-abl fusion protein tyrosine kinase oncogene has been implicated in the pathogenesis of chronic granulocytic leukemia (CGL). Specific intracellular functions performed by p210 bcr-abl have recently been delineated. We considered the possibility that p210 bcr-abl may also regulate the abundance of inosine 5'-monophosphate dehydrogenase (IMPDH) which is a rate-limiting enzyme for de novo guanylate synthesis. We performed studies of the inhibition of IMPDH by tiazofurin, which acts as a competitive inhibitor through its active species that mimics nicotinamide adenine dinucleotide (NAD), i.e. thiazole-4-carboxamide adenine dinucleotide (TAD). The mean inhibitory concentration (IC50) of tiazofurin for cellular proliferation inhibition was 2.3-2.8-fold greater in cells expressing p210 bcr-abl than in their corresponding parent cells proliferating under the influence of growth factors or in growth factor-independent derivative cells not expressing detectable p210 bcr-abl. IMPDH activity was 1.5-2.3-fold greater within cells expressing p210 bcr-abl than in their parent cells. This increase in enzyme activity was a result of 2-fold increased IMPDH protein as determined by immunoblotting. In addition, an increase in the Km value for NAD utilization by IMPDH was observed in p210 bcr-abl transformed cells, but this increase was within the range of resident NAD concentrations observed in the cells. Increased IMPDH protein in p210 bcr-abl transformed cells was traced to an increased level of IMP dehydrogenase II messenger RNA. Thus, regulation of IMPDH gene expression is mediated at least in part by the bcr-abl gene product and may therefore be indicative of a specific mechanism of intrinsic resistance to tiazofurin.

Animals↗

PubMed1994

Granulocyte-macrophage colony-stimulating factor expression is regulated at transcriptional and posttranscriptional levels in a murine bone marrow stromal cell line.

We have reported modulation, by cytokines interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF-alpha) and by hormonal cyclic-adenosine-monophosphate (cAMP) agonists, of hematopoietic growth factor production in the murine marrow adherent cell line +/+(-)1.LDA11. Previously, we reported that increased intracellular cAMP levels inhibited bioactive granulocyte-macrophage colony-stimulatory factor (GM-CSF) production stimulated by IL-1 or by the synergistic stimulus of IL-1 plus TNF-alpha. On the other hand, increased intracellular cAMP stimulated IL-6 synthesis in +/+(-)1.LDA11 cells. In addition, cAMP was additive with either IL-1 or IL-1 plus TNF-alpha in inducing production of soluble IL-6. In the present study, these observations were pursued mechanistically at the level of messenger RNA (mRNA) production. Northern blot analysis of steady-state mRNA for GM-CSF revealed induction by treatment of +/+(-)1.LDA11 cells with IL-1 or with TNF-alpha. The combined stimulation by IL-1 plus TNF-alpha resulted in supra-additive increases in GM-CSF expression by +/+(-)1.LDA11. Addition to stromal cells of the soluble cAMP agonist 8-bromo-cAMP (8BrcAMP) at 0.5 to 1 mM stimulated IL-6 mRNA expression acting alone, and it was additive with IL-1 or IL-1 plus TNF-alpha in stimulating IL-6 expression. On the other hand, 8BrcAMP inhibited GM-CSF mRNA expression stimulated by IL-1 or IL-1 plus TNF-alpha. Inhibition of GM-CSF mRNA by 8BrcAMP was time-dependent, starting 120 to 180 minutes posttreatment. In addition, inhibition of GM-CSF transcript expression in +/+(-)1.LDA11 by 8BrcAMP required the expression of a labile protein. Nuclear run-on assays revealed that GM-CSF and IL-6 genes were transcriptionally induced in +/+(-)1.LDA11 by incubation with IL-1 plus TNF-alpha. IL-6 transcription was further enhanced by 8BrcAMP co-incubation. More sensitive experiments using a luciferase reporter vector containing the GM-CSF promoter region were necessary to convincingly establish the role of TNF-alpha and 8BrcAMP on transcriptional induction of the GM-CSF gene in +/+(-)1.LDA11 stromal cells. Considering these results and an effect of 8BrcAMP on decreasing GM-CSF transcript stability in actinomycin-D (act-D) decay experiments, we conclude that the inhibitory effect of 8BrcAMP on GM-CSF expression is exerted at the posttranscriptional level. These data demonstrate that the intracellular level of cAMP has an important discriminatory role on expression of the cytokines GM-CSF and IL-6 in a model stromal cell line.

Animals↗

PubMed1994

Regulation of interleukin-1 and tumor necrosis factor-alpha induced granulocyte-macrophage colony-stimulating factor gene expression: potential involvement of arachidonic acid metabolism.

Signal transduction pathways evoked by interleukin-1 (IL-1) and tumor necrosis factor-alpha (TNF-alpha) to stimulate expression of other cytokines in mesenchymal cells are not clearly understood. Stimulation of the murine bone marrow stromal cell line +/(+)-1.LDA 11 with IL-1 (500 U/ml) in combination with TNF-alpha (500 U/ml) (IL-1 plus TNF-alpha) induced expression of c-jun mRNA as well as granulocyte-macrophage colony stimulating factor (GM-CSF) mRNA. We investigated the possibility that arachidonic acid metabolites, acting through protein kinase C (PKC) and perhaps also through the PKC-responsive transcription factor c-jun/AP-1, may be responsible for regulating GM-CSF transcription in these stromal cells. Expression of GM-CSF mRNA was preceded by IL-1 plus TNF-alpha induced arachidonate release (assayed using the 3H-derivative). Pretreatment of cells with the phospholipase A2 inhibitor quinacrine (20 microM) inhibited accumulation of both c-jun and GM-CSF mRNA but had no influence on expression of other genes induced by IL-1 and TNF-alpha, including leukemia inhibitory factor (LIF). In addition, quinacrine partially blocked IL-1 plus TNF-alpha induced 3H-arachidonic acid release from prelabeled stromal cells. Furthermore, exogenous arachidonate (10 to 50 microM) induced expression of c-jun. To investigate the role of arachidonate in GM-CSF transcription, we used a reporter vector consisting of the murine GM-CSF promoter linked to firefly luciferase. Transfection efficiency was monitored by assessing expression of a constitutively active gene, RSV-beta galactosidase. In this system, quinacrine significantly inhibited IL-1 plus TNF-alpha induced GM-CSF transcription assayed with the reporter construct. Exogenous arachidonic acid alone (10 microM) increased activity of GM-CSF reporter vector 1.5-fold over control. These results are consistent with the hypothesis that arachidonate metabolites are involved in the signaling pathway that leads to IL-1 plus TNF-alpha induced GM-CSF gene expression. Thus, transcriptional activation of GM-CSF gene is mediated, in part, by the arachidonate cascade.

Animals↗

PubMed1993

Role of p21 RAS in p210 bcr-abl transformation of murine myeloid cells.

The p21 RAS product has been implicated as part of the downstream signaling of certain nonreceptor tyrosine kinase oncogenes and several growth factor receptor-ligand interactions. We have reported that the chronic myelogenous leukemia oncogene p210 bcr-abl transforms a growth-factor-dependent myeloid cell line NFS/N1.H7 to interleukin-3 (IL-3) independence. In these p210 bcr-abl-transformed cells (H7 bcr-abl.A54) and in two other murine myeloid cell lines transformed to IL-3 independence by p210 bcr-abl, endogenous p21 RAS is activated as determined by an elevated ratio of associated guanosine triphosphate (GTP)/guanosine diphosphate (GDP), assayed by thin-layer chromatography of the nucleotides eluted from p21 RAS after immunoprecipitation with the Y13-259 antibody. Treatment of p210 bcr-abl-transformed cells with a specific tyrosine kinase inhibitor herbimycin A resulted in diminished tyrosine phosphorylation of p210 bcr-abl and associated proteins, without major reduction in expression of the p210 bcr-abl protein itself. Inhibition of p210 bcr-abl-dependent tyrosine phosphorylation resulted in a reduction of active p21RAS-GTP complexes in the transformed cells, in diminished expression of the nuclear early response genes c-jun and c-fos, and in lower cellular proliferation rate. To further implicate p21 RAS in these functional events downstream of p210 bcr-abl tyrosine phosphorylation, we targeted G-protein function directly by limiting the availability of GTP with the inosine monophosphate dehydrogenase inhibitor, tiazofurin (TR). In p210 bcr-abl-transformed cells treated for 4 hours with TR, in which the levels of GTP were reduced by 50%, but GDP, guanosine monophosphate, and adenosine triphosphate (ATP) were unaffected, p210 bcr-abl tyrosine phosphorylation was at control levels. However, expression of c-fos and c-jun nuclear proto-oncogenes were strongly inhibited and p21 RAS activity was downregulated. These findings show that p210 bcr-abl transduces proliferative signals, in part, through downstream activation of p21 RAS. Furthermore, p21 RAS activity is linked to pathways that regulate c-jun and c-fos expression.

Animals↗

PubMed1993

Stimulation of interleukin-6 production by either calcitonin gene-related peptide or parathyroid hormone in two phenotypically distinct bone marrow-derived murine stromal cell lines.

It was recently shown that interleukin-6 (IL-6) is produced by bone and bone marrow-derived stromal cells and that it plays an important role in osteoclast development. Here we examined whether parathyroid hormone (PTH), calcitonin (CT), or the calcitonin gene-related peptide (CGRP) influence IL-6 production by two murine bone marrow-derived stromal cell lines: the preadipocyte-like stromal cell line +/+ LDA11 and the fibroendothelial stromal cell line MBA 13.2. We found that CGRP (but not PTH or CT) exerted a dose-dependent increase in cAMP and IL-6 production in the +/+ LDA11 cells. In addition, CGRP had an inhibiting effect on the proliferation of this stromal cell line. CGRP, however, did not affect cAMP or IL-6 in the rat osteogenic sarcoma cell line UMR-106-06, which exhibits CT receptors, whereas CT stimulated both cAMP and IL-6 by the UMR-106-01 cells. In contrast to the specificity of the IL-6 response of the +/+ LDA11 cells to CGRP, IL-6 production by the MBA 13.2 stromal cells was stimulated by PTH whereas CGRP or CT had no effect. These data suggest that bone marrow-derived stromal cells express receptors for either CGRP or PTH in a phenotype-specific manner and that, acting via these receptors, CGRP and PTH stimulate IL-6 production by stromal cells. In addition, the evidence for specific receptors for the neuropeptide CGRP in bone marrow stromal cells and an effect of CGRP on IL-6 raises the possibility for a role of cytokines in a putative interplay between neuronal stimuli and bone.

Animals↗

PubMed1993

LIF mRNA expression is transcriptionally regulated in murine bone marrow stromal cells.

Recent evidence has established an important role for leukemia inhibitory factor (LIF) as hematopoietically active cytokine. The present study utilized two different murine bone marrow stromal cell lines, +/+-1.LDA11 and MBA-13, to define regulatory mechanisms of LIF messenger RNA (mRNA) induction. LIF mRNA was not detected in uninduced stromal cells under serum-free conditions. Incubation with interleukin-1 (IL-1), tumor necrosis factor-alpha (TNF-alpha) or the cAMP analogue 8-bromoadenosine 3':5'-monophosphate (8BrcAMP) resulted in weakly induced LIF mRNA. Coincubation of combinations of the stimuli increased LIF mRNA expression additively. LIF mRNA stability, even after stimulation, was low with a half-life of about 30 min, suggesting a functional role for known AU-rich motifs in the 3' untranslated LIF mRNA region in mediating this instability. This possibility was further supported by the ability of cycloheximide to increase mRNA levels without affecting transcription. Transcriptional activation was found to be the main mechanism leading to LIF mRNA expression by IL-1, by TNF-alpha, and by 8BrcAMP. These stimuli appeared to act additively in this regard, suggesting involvement of distinct transcription factors. Induction of transcription was detected 45 min post-stimulation and showed peak levels at 90 min. Kinetics of LIF transcriptional activation showed similarity with the kinetics of the transcription factors, jun-B and c-fos, suggesting a possible role for these proteins or other early response genes in events leading to LIF expression.

8-Bromo Cyclic Adenosine Monophosphate↗