Abstract: Human breast cancer (HBC) cell growth suppression by okadaic acid (OA) was previously found to involve elevated expression of oncogenes c-myc and c-fos and apoptosis. Since, c-Myc influences diverse pathways of cell growth, we hypothesized that elevated levels of c-Myc are involved in HBC growth suppression. Here, we investigated whether induction of c-Myc by OA or protein synthesis inhibitor cycloheximide contributed to HBC growth inhibition and the mechanisms involved. OA, cycloheximide, or the chemotherapeutic drug Taxol suppressed HBC cell growth. However, OA or cycloheximide treatments over 6 or 10 h, respectively, induced c-Myc expression. Depletion of c-Myc, on the other hand, resulted in enhanced HBC cell viabilities when exposed to OA or cycloheximide, but not by Taxol. OA induced c-myc transcription by targeting an 80-bp region from positions -11 to +70, relative to the P1 transcription start of mouse c-myc promoter. Gel mobility shift assays revealed binding of HBC cell nuclear proteins to the OA-responsive c-myc promoter fragment, whereas binding of one complex was elevated in the case of the OA-treated or cycloheximide-treated HBC cell nuclear extracts. Database search revealed presence of a consensus sequence for zinc finger protein gut-enriched Kruppel-like factor (GKLF) in OA-responsive region of the c-myc promoter. Mutation of GKLF consensus sequences abrogated OA responsiveness of the c-myc promoter, and OA treatments caused enhanced expression of GKLF in HBC cells. Thus, OA-dependent attenuation of HBC growth is accomplished, in part, by zinc finger transcription factor GKLF-mediated enhanced transcription of c-myc.

Abstract: Macrophages play an important role in immune responses and in inflammatory disease states such as atherosclerosis. Interferon-gamma (IFN-gamma) is a major cytokine involved in the activation of macrophages. To elucidate the primary response of various genes and biological pathways regulated by IFN-gamma in macrophage, we analyzed the gene expression profile in RAW 264.7 macrophage cells treated with IFN-gamma for 4h. Microarray analysis revealed that about 400 genes were differentially expressed, of which about 250 genes were up-regulated and 150 were down-regulated. Functional organization of the transcriptome revealed that induced genes are involved in antimicrobial and antiviral responses, antigen presentation, chemokine and cytokine signaling, and inhibition of cell growth. We also found that expression of genes involved in cell-cycle control, DNA repair, and lipid metabolism was suppressed by IFN-gamma. We also identified induction of multiple transcription factors by IFN-gamma in RAW 264.7 cells. Functional annotation of genes regulated by IFN-gamma in RAW 264.7 cells may provide novel insights into the role of macrophages in immunity and in inflammatory disease.

Abstract: CD8+ T cell recognition of viral antigens presented by lung epithelial cells is important in the clearance of respiratory viral infection but may cause considerable injury to the lung. We have shown that a critical event of this type of injury is the activation of target epithelial cells and expression of chemokines by these cells. In this study, epithelial gene expression and transcription factor activation triggered by specific CD8+ T cell antigen recognition was examined in vitro and in vivo. T cell recognition triggers expression profiles of tumor necrosis factor-alpha (TNF-alpha)-dependent and interferon-gamma (IFN-gamma)-dependent genes in epithelial target cells. Consistent with these profiles, transcription factors nuclear factor-kappaB (NF-kappaB) and activator protein-1 (AP-1) were activated in lung epithelial cells of wild-type (WT) mice but not TNF receptor 1 (TNFR1)-deficient mice after CD8+ T cell recognition in vivo. In contrast, Stat1 activation and Stat1-dependent genes, such as IFN regulatory factor-1 (IRF-1) and guanylate-binding protein-2 (GBP-2), were induced to a similar extent in epithelial cells of both WT and TNFR1-deficient mice, indicating that this pathway is insufficient to induce pulmonary immunopathology in the absence of NF-kappaB-dependent transcriptional activation. Antibody neutralization of TNF-alpha abrogated epithelial monocyte chemotactic protein-1 (MCP-1) and macrophage inflammatory protein-2 (MIP-2) production in vitro as well as pulmonary immunopathology in vivo, confirming the primary importance of this cytokine in CD8+ T cell-mediated immunopathology.

Abstract: Lipoprotein lipase (LPL) is a key enzyme in the hydrolysis of triglyceride-rich lipoproteins. In vascular diseases, such as atherosclerosis, inflammation plays an important role in the pathogenesis of the disease. We examined the role of LPL in modulating tumor necrosis factor-alpha (TNF-alpha)- and interferon-gamma (IFN-gamma)-mediated inflammatory cytokine signal transduction pathways in human aortic endothelial cells (HAECs). LPL significantly suppressed TNF-alpha-induced gene expression, and this suppression was reversed by tetrahydrolipstatin and heparinase. In contrast, LPL synergistically enhanced IFN-gamma-induced gene expression in HAECs. To elucidate the molecular mechanisms of LPL action, we investigated the role of transcription factors nuclear factor kappa B (NF-kappaB) and signal transducer and activator of transcription factor 1 (Stat1). The anti-inflammatory response of LPL in suppressing TNF-alpha-induced gene expression was a result of its inhibition of NF-kappaB activity by the abrogation of IkappaB-alpha degradation and phosphorylation of the p65 subunit. Although LPL alone had no effect on Stat1 activation, LPL enhanced IFN-gamma-induced phosphorylation of Stat1 on tyrosine 701 and serine 727, as well as Stat1-mediated transactivation. The synergistic effect of LPL on IFN-gamma-induced Stat1 activation was mediated by enhanced activation of the tyrosine kinase JAK2 and was abrogated by LY294002, a specific inhibitor of the phosphatidylinositol 3'-kinase pathway. Our studies indicate that LPL has differential effects on several inflammatory pathways known to be important in atherosclerosis.

Abstract: Using microarray technology, we previously demonstrated that IFN-gamma induces suppressor of cytokine signaling-3 (SOCS-3) in Stat1-/- mouse embryonic fibroblasts and bone marrow-derived macrophages. In this study, we have investigated the mechanism by which SOCS-3 is induced by Stat1-independent signal transduction pathway. Tyrosine kinases Jak1 and Jak2 are required for SOCS-3 induction by IFN-gamma in mouse embryonic fibroblasts. IFN-gamma stimulated strong and sustained activation of Stat1 whereas Stat3 activation was weak and transient in wild-type fibroblasts. In contrast, Stat3 is activated strongly and in a sustained manner in Stat1-/- fibroblasts. The Src kinase inhibitor SU6656 suppressed IFN-gamma activation of Stat3 in both wild-type and Stat1-/- fibroblasts. However, SU6656 inhibited IFN-gamma induction of SOCS-3 completely in Stat1-/- but not in wild-type fibroblasts. Knock down of Stat3 by short interfering RNA abrogated Stat3 activation and SOCS-3 induction by IFN-gamma in Stat1-/- fibroblasts. In human fibrosarcoma cell line 2fTGH, IFN-gamma activated Stat1 but not Stat3. SOCS-3 induction by IFN-gamma is strictly Stat1-dependent. The Stat1 docking site is required for SOCS-3 induction by IFN-gamma in human lung adenocarcinoma cells. We propose a model in which sustained activation of Stat1 or Stat3 mediates SOCS-3 induction by IFN-gamma in wild-type and Stat1-/- mouse embryonic fibroblasts, respectively.

Abstract: Respiratory virus infection evokes a potent T-cell response that may result in a considerable insult to the structural and functional integrity of the gas exchange units of the lung. Alveolar antigen recognition by CD8+ T lymphocytes results in significant injury that is critically dependent upon tumor necrosis factor (TNF)-alpha expressed by the CD8+ T cells and is largely dependent upon TNF-receptor 1 expression on the alveolar epithelial target cells. TNF-receptor 2 (TNF-R2)-deficient mice were used to demonstrate that CD8+ T-cell-mediated lung injury associated with clearance of experimental influenza requires TNF-R2 for full expression of immunopathology. In vitro analysis indicates that alveolar cell expression of TNF-R2 is critical in the induction of epithelial monocyte chemoattractant protein (MCP)-1 expression specifically in response to soluble TNF-alpha, suggesting an important role for this receptor in bystander lung injury. However, TNF-R2 was dispensable for induction of alveolar MCP-1 expression in response to transmembrane TNF-alpha expressed by antigen-specific CD8+ T cells, and the effects of the two receptors seem to be additive. Because TNF-R2 may be rapidly shed as part of feedback inhibition of bystander inflammation, this suggests a mechanism by which immunopathology in respiratory virus infection may be regulated and by which T-cell receptor-dependent TNF-alpha activity might bypass such negative regulation for contact-dependent antiviral activities.

Abstract: p53 has been well characterized as a tumor suppressor gene, but its role in antiviral defense remains unclear. A recent report has demonstrated that p53 can be induced by interferons and is activated after vesicular stomatitis virus (VSV) infection. We observed that different nononcogenic viruses, including encephalomyocarditis virus (EMCV) and human parainfluenza virus type 3 (HPIV3), induced down-regulation of p53 in infected cells. Double-stranded RNA (dsRNA) and a mutant vaccinia virus lacking the dsRNA binding protein E3L can also induce this effect, indicating that dsRNA formed during viral infection is likely the trigger for down-regulation of p53. The mechanism of down-regulation of p53 by dsRNA relies on translation inhibition mediated by the PKR and RNase L pathways. In the absence of p53, the replication of both EMCV and HPIV3 was retarded, whereas, conversely, VSV replication was enhanced. Cell cycle analysis indicated that wild-type (WT) but not p53 knockout (KO) fibroblasts undergo an early-G(1) arrest following dsRNA treatment. Moreover, in WT cells the onset of dsRNA-induced apoptosis begins after p53 levels are down-regulated, whereas p53 KO cells, which lack the early-G(1) arrest, rapidly undergo apoptosis. Hence, our data suggest that the down-regulation of p53 facilitates apoptosis, thereby limiting viral replication.

Abstract: Proinflammatory cytokines have been linked to depression of myocardial contractility in vivo in patients with acute septic shock and in vitro models employing isolated myocytes exposed to serum from such patients. The key pathways involved in mediating this septic organ dysfunction (cell adhesion molecule expression, inducible nitric-oxide synthase induction, and apoptosis) are known to be regulated by transcription factors STAT1, IRF1, and NF-kappaB. Utilizing a model that mimics human disease, we have demonstrated activation of the transcription factors STAT1, IRF1, and NF-kappaB in human fetal myocytes exposed to human septic serum. Both reporter and electrophoretic mobility shift assays demonstrated a 5-19-fold increase in activation of transcription factors STAT1, IRF1, and NF-kappaB in response to incubation with human septic serum. The addition of human septic serum to human fetal myocytes induced apoptosis in human fetal myocytes and activation of the mitogen-activated protein kinase c-Jun NH -terminal kinase and caspase 1 as measured by Western blot. These data suggest that transcription factor activation and early myocyte apoptosis play a mechanistic role in septic myocardial depression and sepsis-induced organ dysfunction.

Abstract: Respiratory virus infection results in considerable pulmonary immunopathology, a component of which results from the host immune responses. We have developed a murine model to specifically examine the lung injury due to CD8(+) T cell recognition of an influenza hemagglutinin (HA) transgene on lung epithelium in the absence of replicating virus, after adoptive transfer. Lung injury is largely mediated by chemokines expressed by the epithelial cells upon T cell recognition mediated by TNF-alpha. To determine the critical source of TNF-alpha, HA-specific TNF(-/-) CD8(+) T cells were transferred into HA transgenic animals, and lung injury was not observed, though these T cells exhibited no defect in antiviral activity in vivo. This indicates that the initiating event in the injury process is Ag-specific expression of TNF-alpha by antiviral CD8(+) T cells upon recognition of alveolar epithelial Ag, and that the effector activities responsible for viral clearance may be dissociable from those resulting in immunopathology.

Abstract: Interleukin-1 (IL-1) induces the phosphorylation of Stat1 on serine 727 but not on tyrosine 701. Analyses of mutant I1A cells, which lack the IL-1 receptor-associated kinase (IRAK), and of I1A cells reconstituted with deletion mutants of IRAK show that the IL-1-mediated phosphorylation of Stat1 on serine requires the IRAK protein but not its kinase activity and does not involve phosphatidylinositol-3'-kinase (PI3K) or the mitogen-activated protein (MAP) kinases p38 or ERK. IRAK and Stat1 interact in vivo, and this interaction is increased in response to IL-1, suggesting that IRAK may serve to recruit the as yet unknown IL-1-induced Stat1 serine kinase. Chemical inhibitors or dominant-negative forms of signaling components required to activate NF-kappa B, ATF, or AP-1 in response to IL-1 do not affect the phosphorylation of Stat1 on serine. IL-1 and tumor necrosis factor (TNF) enhance the serine phosphorylation of Stat1 that occurs in response to interferon-gamma (IFN-gamma) and potentiate IFN-gamma-mediated, Stat1-driven gene expression, thus contributing to the synergistic activities of these proinflammatory cytokines.

Abstract: The cytokine gamma interferon (IFN-gamma) and the calcitropic steroid hormone 1,25-dihydroxyvitamin D (1,25D) are activators of macrophage immune function. In sarcoidosis, tuberculosis, and several granulomatoses, IFN-gamma induces 1,25D synthesis by macrophages and inhibits 1,25D induction of 24-hydroxylase, a key enzyme in 1,25D inactivation, causing high levels of 1,25D in serum and hypercalcemia. This study delineates IFN-gamma-1,25D cross talk in human monocytes-macrophages. Nuclear accumulation of Stat1 and vitamin D receptor (VDR) by IFN-gamma and 1,25D promotes protein-protein interactions between Stat1 and the DNA binding domain of the VDR. This prevents VDR-retinoid X receptor (RXR) binding to the vitamin D-responsive element, thus diverting the VDR from its normal genomic target on the 24-hydroxylase promoter and antagonizing 1,25D-VDR transactivation of this gene. In contrast, 1,25D enhances IFN-gamma action. Stat1-VDR interactions, by preventing Stat1 deactivation by tyrosine dephosphorylation, cooperate with IFN-gamma/Stat1-induced transcription. This novel 1,25D-IFN-gamma cross talk explains the pathogenesis of abnormal 1,25D homeostasis in granulomatous processes and provides new insights into 1,25D immunomodulatory properties.

Abstract: The 90-kDa heat shock protein (Hsp90), the target of the ansamycin class of anti-cancer drugs, is required for the conformational activation of a specific group of signal transducers, including Raf-1. In this report we have identified a 75-kDa Raf-associated protein as Hsp90N, a novel member of the Hsp90 family. Intriguingly, the ansamycin-binding domain is replaced in Hsp90N by a much shorter, hydrophobic sequence, preceded by a putative myristylation signal. We demonstrate that, although much less abundant, Hsp90N binds Raf with a higher affinity than Hsp90. In sharp contrast to Hsp90, Hsp90N does not associate with p50(cdc37), the Hsp90 kinase cofactor. Hsp90N was found to activate Raf in transiently transfected cells, while Rat F111 fibroblasts stably transfected with Hsp90N exhibited elevated activity of the Raf and downstream ERK kinases. This may be due to Raf binding to myristylated Hsp90N, followed by Raf translocation to the membrane. To examine whether Hsp90N could therefore substitute for Ras in Raf recruitment to the cell membrane, Hsp90N was transfected in c-Ras-deficient, 10T1/2-derived preadipocytes. Our results indicate that, as shown before for activated Ras or Raf, the introduction of even low levels of Hsp90N through transfection in c-Ras-deficient preadipocytes causes a dramatic block of differentiation. Higher levels of Hsp90N expression resulted in neoplastic transformation, including interruption of gap junctional, intercellular communication, and anchorage-independent proliferation. These results indicate that the observed activation of Raf by Hsp90N has a profound biological effect, which is largely c-Ras-independent. With the recent finding that p50(cdc37) is tumorigenic in transgenic mice, these results reinforce the intriguing observation that the family of heat shock proteins represents a novel class of molecules with oncogenic potential.

Abstract: The paradigm that emerged from studies during the past decade established a central role for Jak-Stat (signal transducer and activator of transcription) signaling pathways in promoting the diverse cellular responses induced by interferon gamma (IFN-gamma). However, recent studies have shown that the IFN-gamma receptor activates additional signaling pathways and can regulate gene expression by Stat1-independent pathways. The diversity of gene-expression patterns mediated by Stat1-dependent and -independent mechanisms and the balance between these two pathways play an important role in the biological response to IFN-gamma.

Abstract: Although Stat1 is essential for cells to respond fully to IFN-gamma, there is substantial evidence that, in the absence of Stat1, IFN-gamma can still regulate the expression of some genes, induce an antiviral state and affect cell growth. We have now identified many genes that are regulated by IFN-gamma in serum-starved Stat1-null mouse fibroblasts. The proteins induced by IFN-gamma in Stat1-null cells can account for the substantial biological responses that remain. Some genes are induced in both wild-type and Stat1-null cells and thus are truly Stat1-independent. Others are subject to more complex regulation in response to IFN-gamma, repressed by Stat1 in wild-type cells and activated in Stat1-null cells. Many genes induced by IFN-gamma in Stat1-null fibroblasts also are induced by platelet-derived growth factor in wild-type cells and thus are likely to be involved in cell proliferation. In mouse cells expressing the docking site mutant Y440F of human IFN-gamma receptor subunit 1, the mouse Stat1 is not phosphorylated in response to human IFN-gamma, but c-myc and c-jun are still induced, showing that the Stat1 docking site is not required for Stat1-independent signaling.

Abstract: Although Stat1 is required for many IFN-dependent responses, recent work has shown that IFNgamma functions independently of Stat1 to affect the growth of tumor cells or immortalized fibroblasts. We now demonstrate that both IFNgamma and IFNalpha/beta regulate proliferative responses in cells of the mononuclear phagocyte lineage derived from Stat1-null mice. Using both representational difference analysis and gene arrays, we show that IFNgamma exerts its Stat1-independent actions on mononuclear phagocytes by regulating the expression of many genes. This result was confirmed by monitoring changes in expression and function of the corresponding gene products. Regulation of the expression of these genes requires the IFNgamma receptor and Jak1. The physiologic relevance of IFN-dependent, Stat1-independent signaling was demonstrated by monitoring antiviral responses in Stat1-null mice. Thus, the IFN receptors engage alternative Stat1-independent signaling pathways that have important physiological consequences.

Abstract: STAT1 must be phosphorylated on serine 727 to be fully active in transcription. We show that phosphatidylinositol 3-kinase (PI3K) and its effector kinase Akt play an important role in the serine phosphorylation of STAT1 and in the activation of gene expression in response to interferon-gamma (IFN gamma). IFN gamma activates PI3K as well as Akt in a variety of cell lines. Specific inhibition of PI3K abrogates IFN gamma-induced, but not interleukin-1- or tumor necrosis factor-alpha-induced, phosphorylation of STAT1 on serine and reduces STAT1-dependent transcription and gene expression by approximately 7-fold. Constitutively active forms of PI3K or Akt activate and their dominant-negative derivatives inhibit STAT1-driven transactivation in response to IFN gamma. In addition to PI3K and Akt, JAK1, JAK2, and the tyrosine 440 STAT1 docking residue of IFNGR1 are required for STAT1 to be phosphorylated on serine. Taken together, these results suggest that the following events lead to the activation of STAT1 upon IFN gamma stimulation: 1) PI3K and Akt are activated by the occupied receptor and Tyr-440 is phosphorylated by the activated JAKs; 2) STAT1 docks to Tyr-440; and 3) Tyr-701 is phosphorylated by the JAKs and Ser-727 is phosphorylated by a kinase downstream of Akt.

Abstract: Activation of MAP kinase leads to the activation of p53-dependent pathways, and vice-versa. Although the amount of p53 protein increases in response to MAP kinase-dependent signaling, the basis of this increase is not yet fully understood. We have isolated the mutant cell line AP14, defective in p53 expression, from human HT1080 fibrosarcoma cells, which have an activated ras allele. The expression of p53 mRNA and protein is approximately 10-fold lower in AP14 cells than in the parental cells. The high constitutive phosphorylation and activities of the MAP kinases ERK1 and ERK2 in HT1080 cells are greatly reduced in AP14 cells, although the levels of these proteins are unchanged, suggesting that the defect in the mutant cells affects the steady-state phosphorylation of ERK1 and ERK2. Overexpression of ERK2 in AP14 cells restored both MAP kinase activity and p53 expression, and incubation of the mutant cells with the phosphatase inhibitor orthovanadate resulted in strong coordinate elevation of MAP kinase activity and p53 expression. The levels of expression of the p53-regulated gene p21 parallel those of p53 throughout, showing that basal p21 expression depends on p53. The levels of p53 mRNA increased by 5-8-fold when activated ras was introduced into wild-type cells, and the levels of the p53 and p21 proteins decreased substantially in wild-type cells treated with the MEK inhibitor U0216. We conclude that MAP kinase-dependent pathways help to regulate p53 levels by regulating the expression of p53 mRNA.

Abstract: Stat1 is a fascinating and complex protein with multiple, yet contrasting transcriptional functions. Upon activation, it drives the expression of many genes but also suppresses the transcription of others. These opposing characteristics also apply to its role in facilitating crosstalk between signal transduction pathways, as it participates in both synergistic activation and inhibition of gene expression. Stat1 is a functional transcription factor even in the absence of inducer-mediated activation, participating in the constitutive expression of some genes. This review summarizes the well studied involvement of Stat1 in IFN-dependent and growth factor-dependent signaling and then describes the roles of Stat1 in positive, negative and constitutive regulation of gene expression as well as its participation in crosstalk between signal transduction pathways. Oncogene (2000).

Abstract: Interferons (IFNs) inhibit cell growth in a Stat1-dependent fashion that involves regulation of c-myc expression. IFN-gamma suppresses c-myc in wild-type mouse embryo fibroblasts, but not in Stat1-null cells, where IFNs induce c-myc mRNA rapidly and transiently, thus revealing a novel signaling pathway. Both tyrosine and serine phosphorylation of Stat1 are required for suppression. Induced expression of c-myc is likely to contribute to the proliferation of Stat1-null cells in response to IFNs. IFNs also suppress platelet-derived growth factor (PDGF)-induced c-myc expression in wild-type but not in Stat1-null cells. A gamma-activated sequence element in the promoter is necessary but not sufficient to suppress c-myc expression in wild-type cells. In PKR-null cells, the phosphorylation of Stat1 on Ser727 and transactivation are both defective, and c-myc mRNA is induced, not suppressed, in response to IFN-gamma. A role for Raf-1 in the Stat1-independent pathway is revealed by studies with geldanamycin, an HSP90-specific inhibitor, and by expression of a mutant of p50(cdc37) that is unable to recruit HSP90 to the Raf-1 complex. Both agents abrogated the IFN-gamma-dependent induction of c-myc expression in Stat1-null cells.

Abstract: O6-methylguanine-DNA methyltransferase (MGMT), a ubiquitous DNA repair protein, removes the mutagenic DNA adduct O6-alkylguanine, which is synthesized both endogenously and after exposure to alkylnitrosamines and alkylating antitumor drugs such as 2-chloroethyl-N-nitrosourea (CNU). The MGMT gene is highly regulated in mammalian cells and its overexpression, observed in many types of tumor cells, is often associated with cellular resistance to CNU. Dexamethasone, a synthetic glucocorticoid hormone, was found to increase MGMT expression in HeLa S3 cells, concomitant with their increased resistance to CNU. Two putative glucocorticoid responsive elements (GREs) were identified in the human MGMT (hMGMT) promoter. Transient expression of the luciferase reporter gene driven by an hMGMT promoter fragment containing these GREs was activated by dexamethasone. DNase I footprinting assays demonstrated the binding of glucocorticoid receptor to these sequences. In vitro transcription experiment showed that these DNA sequences are functional in glucocorticoid receptor signal-mediated activation of transcription. These results suggest glucocorticoid-mediated induction of the MGMT gene contributes to high level expression of MGMT.

Abstract: Treatment of mouse or human cells with the protein kinase C (PKC) inhibitors H7 or bisindolylmaleimide I induced an increase in the lifetime of p53, leading to its accumulation. In inhibitor-treated cells, p53 translocated to the nuclei and bound to DNA but was not competent to induce transcription. However, transactivation could be induced by subsequent DNA damage. Phorbol ester, a potent activator of PKC, significantly inhibited the accumulation of p53 after DNA damage. Therefore, constitutive PKC-dependent phosphorylation of p53 itself, or of a protein that interacts with p53, is required for the rapid degradation of p53 in untreated cells. Furthermore, an increase in the lifetime of p53 is not accompanied necessarily by its activation. Treatment with the PKC inhibitors decreased the overall level of p53 phosphorylation but led to the appearance of a phosphopeptide not seen in tryptic digests of p53 from untreated cells. Therefore, the lifetime and activities of p53 are likely to be regulated by distinct alterations of the phosphorylation pattern of p53, probably caused by the actions of different kinases.

Abstract: Apurinic/apyrimidinic (AP) endonuclease (APE; EC 4.2.99.18) plays a central role in repair of DNA damage due to reactive oxygen species (ROS) because its DNA 3'-phosphoesterase activity removes 3' blocking groups in DNA that are generated by DNA glycosylase/AP-lyases during removal of oxidized bases and by direct ROS reaction with DNA. The major human APE (APE-1) gene is activated selectively by sublethal levels of a variety of ROS and ROS generators, including ionizing radiation, but not by other genotoxicants-e.g., UV light and alkylating agents. Increased expression of APE mRNA and protein was observed both in the HeLa S3 tumor line and in WI 38 primary fibroblasts, and it was accompanied by translocation of the endonuclease to the nucleus. ROS-treated cells showed a significant increase in resistance to the cytotoxicity of such ROS generators as H2O2 and bleomycin, but not to UV light. This "adaptive response" appears to result from enhanced repair of cytotoxic DNA lesions due to an increased activity of APE-1, which may be limiting in the base excision repair process for ROS-induced toxic lesions.

Abstract: O6-Alkylguanine is the major mutagenic and cytotoxic DNA lesion induced by alkylating agents, including 2-chloroethyl-N-nitrosourea-based antitumor drugs. This lesion is repaired by O6-methylguanine-DNA methyltransferase (MGMT), the expression of which is highly variable in both normal tissues and in tumor cells. The promoter of the human MGMT gene was found to contain two putative activator protein (AP)-1 sites. Here, we show that the level of MGMT mRNA in HeLa S3 cells was increased 3-5-fold by phorbol-12-myristate-13-acetate (TPA) and 1,2-diacyl-sn-glycerol (DAG), which are activators of protein kinase C (PKC), as well as by okadaic acid, an inhibitor of protein phosphatases. The PKC inhibitor 1-(5-isoquinoline sulfonyl)-2-methylpiperazine-HCl eliminated MGMT activation by TPA and DAG but not by OA. Prior down-regulation of PKC abolished subsequent effects of TPA or DAG. The results indicate AP-1 to be involved in regulation of MGMT expression. This hypothesis was supported by showing AP-1 binding to two target sequences of the MGMT promoter and transactivation of the MGMT promoter upon cotransfection with c-fos and c-jun in F9 cells. That TPA-mediated induction of MGMT caused increased cellular resistance to 2-chloroethyl-N-nitrosourea suggests a therapeutic significance for PKC-mediated MGMT modulation.

Abstract: N-Methylpurine-DNA glycosylase (MPG), a ubiquitous DNA repair enzyme, removes N-alkylpurines and other purine lesions induced in DNA by simple alkylating carcinogens. A mouse MPG cDNA clone was isolated from a lambda recombinant phage library of BALB/c mouse lung cell and characterized. Using the mouse MPG cDNA as a probe, the complete mouse MPG gene was isolated in two overlapping lambda recombinant genomic clones. The 6-kb gene has four exons containing 1,002 bp of coding sequence. The transcription start site was identified in the genomic sequence by primer extension of MPG mRNA from a mouse lung fibroblast cell line. The location of this transcription start site was confirmed by in vitro transcription with the promoter-containing plasmid template. Promoter function of the sequence 5' upstream of the transcription initiation site was shown by transient expression of the firefly luciferase reporter gene under the control of this sequence in transfected human and mouse cells. The mouse MPG promoter contains no TATA box, but has a CAAT element and is G.C-rich with putative AP2 elements and SP1-complementary sequences.

Abstract: Dorsal root ganglia neuronal cell bodies synthesize the vasodilator neuropeptide calcitonin gene-related peptide and innervate the blood vessels and spinal cord sites (laminae I and II) involved in blood pressure regulation. We previously demonstrated that calcitonin gene-related peptide mRNA content is significantly decreased in dorsal root ganglia and that immunoreactive calcitonin gene-related peptide levels are reduced in laminae I and II of the dorsal horn of the spinal cord in the spontaneously hypertensive rat compared with Wistar-Kyoto control rats. To determine whether neuronal calcitonin gene-related peptide expression is also altered in mineralocorticoid-salt hypertension, we quantified calcitonin gene-related peptide mRNA levels in dorsal root ganglia and protein content in laminae I and II of the spinal cord in rats with mineralocorticoid-salt-induced hypertension. To control for pellet implantation, saline drinking water, and/or uninephrectomy, four normotensive groups were similarly studied. By Northern hybridization analysis, the ratio of calcitonin gene-related peptide mRNA to 18S rRNA was increased approximately fivefold in hypertensive rats (33 +/- 7) compared with each of the four normotensive control groups (average of the four groups, 6 +/- 0.5; P < .01, mineralocorticoid-salt group versus each group). The density of the peptide, quantified by computer-assisted image analysis, in laminae I and II in the hypertensive rats was also increased (66 +/- 1 versus average of the four groups, 46 +/- 2 arbitrary units; P < .001, mineralocorticoid-salt group versus each group). In conclusion, neuronal levels of calcitonin gene-related peptide mRNA and protein are increased in mineralocorticoid-salt hypertension.(ABSTRACT TRUNCATED AT 250 WORDS)

Abstract: Calcitonin gene-related peptide, a product of the calcitonin gene, is a potent vasodilator neuropeptide. We have demonstrated that dietary calcium deficiency decreased the neuronal (laminae I/II of the dorsal horn of the spinal cord) content of immunoreactive calcitonin gene-related peptide in the normal rat. Neuronal calcitonin gene-related peptide levels are also reduced in the spontaneously hypertensive rat, a model characterized by calcium deficiency. However, the mechanism of this reduction in neuronal calcitonin gene-related peptide could be due to decreased synthesis or increased release. To determine if neuronal calcitonin gene-related peptide messenger RNA (mRNA) levels are also decreased in the spontaneously hypertensive rat, we measured relative calcitonin gene-related peptide mRNA levels (using a genomic hybridization probe specific for alpha- and beta-calcitonin gene-related peptide mRNA) in dorsal root ganglia from spontaneously hypertensive and Wistar-Kyoto control rats. Dorsal root ganglia neuronal cell bodies are a prominent site of calcitonin gene-related peptide synthesis and send axons to peripheral blood vessels and central spinal cord sites (laminae I/II). After normalization of calcitonin gene-related peptide mRNA levels of 18S RNA, the calcitonin gene-related peptide mRNA/18S RNA ratio was significantly decreased approximately threefold in the spontaneously hypertensive rats compared with controls. This alteration in calcitonin gene-related peptide mRNA levels is specific for dorsal root ganglia, because no strain differences in calcitonin gene-related peptide mRNA content were detected in heart or brain.(ABSTRACT TRUNCATED AT 250 WORDS)

Abstract: Calcitonin gene-related peptide (CGRP), a product of the calcitonin/CGRP gene, is a potent vasodilating neuropeptide widely distributed throughout the cardiovascular system, particularly in the heart. Immunocytochemical studies have demonstrated CGRP-containing neurofibrils in the myocardium and in the periadventitia of coronary blood vessels. Based on these studies, it has been assumed that all of the CGRP peptide in the heart is synthesized in neurons whose cell bodies are located outside of the heart. Using Northern blot analysis and a ribonuclease protection assay, we observed in the rat heart low levels of a CGRP-like mRNA species that appeared to be identical to authentic CGRP mRNA produced in the brain and dorsal root ganglia. The ventricles contained somewhat more CGRP mRNA than did the atria. Also, whereas the dorsal root ganglia synthesized both alpha- and beta-CGRP mRNA, only the alpha-CGRP mRNA was detected in the heart. The presence of CGRP mRNA in the heart suggests that the CGRP gene is transcriptionally active in a subpopulation of heart cells, possibly neuronal, which have the potential to synthesize and secrete this neuropeptide. Given the potent coronary vasodilatory and positive chronotropic and inotropic effects of CGRP, the localized synthesis of CGRP in the heart may play a role in modulating cardiovascular function.