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PubMed1990

Induction of C-reactive protein by cytokines in human hepatoma cell lines is potentiated by caffeine.

Induction of C-reactive protein (CRP) by conditioned medium from lipopolysaccharide-stimulated human monocytes in two human hepatoma-cell lines, Hep 3B and NPLC/PRF/5, was potentiated 3-6-fold by the methylxanthine caffeine. The induction observed in the presence of conditioned medium plus caffeine was as much as 180-fold, comparable with that seen after many stimuli in vivo. This potentiation was accompanied by an increase in the levels of CRP mRNA. By contrast, no potentiating effect on CRP induction by conditioned medium was found when we tested theophylline, forskolin, 8-bromo cyclic AMP or two Ca2+ ionophores, namely ionomycin and A23187. None of the above compounds, including caffeine, when tested alone, had any detectable effect on the synthesis and secretion of CRP. Our previous study [Ganapathi, May, Schultz, Brabenec, Weinstein, Sehgal & Kushner (1988) Biochem. Biophys. Res. Commun. 157, 271-277], employing defined cytokines, had shown that induction of CRP in Hep 3B cells requires IL(interleukin)-6 plus IL-1, whereas, in the NPLC/PRF/5 cell line, IL-6 alone is effective. Caffeine similarly potentiated induction of CRP by these defined cytokine signals in these two cell lines. Changes in synthesis of other acute-phase proteins, including serum amyloid A (SAA), alpha 1-proteinase inhibitor, alpha 1-antichymotrypsin and albumin, induced by conditioned medium or, in some cases, by IL-6 and/or IL-1 alpha, were only minimally affected by caffeine. Thus these results indicate that the mechanism by which caffeine potentiates CRP induction by cytokines appears to be independent of increases in intracellular concentrations of the two second messengers, cyclic AMP and Ca2+; the precise nature of this mechanism is unclear at the present time. Our results also indicate that the intracellular mechanisms by which cytokines regulate synthesis of CRP may differ from those regulating synthesis of some other acute-phase proteins. The differential response of CRP and SAA to caffeine is of particular interest, since induction of both of these two major acute-phase proteins can be accomplished by identical extracellular signals.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1994

Differential effects of 9-cis and all-trans retinoic acid on the induction of retinoic acid receptor-beta and cellular retinoic acid-binding protein II in human neuroblastoma cells.

The objective of this study was to compare the properties of 9-cis and all-trans retinoic acid with respect to the induction of expression of retinoic acid receptor beta (RAR-beta) and cellular retinoic acid-binding protein (CRABP) II in human neuroblastoma SH SY 5Y cells. RAR-beta and CRABP II mRNA was induced by both all-trans and 9-cis retinoic acid in SH SY 5Y cells. Induction was rapid, detectable within 2-4 h, and inhibited by actinomycin D. Time-courses of induction for RAR-beta and CRABP II differed: RAR-beta mRNA levels reached a maximum 4-6 h after adding all-trans or 9-cis retinoic acid, whereas CRABP II mRNA levels increased over at least 18 h. These differences were attributed to the longer half-life of CRABP II mRNA (20 h) compared with RAR-beta mRNA (3.9 h). The dose-response characteristics of all-trans and 9-cis retinoic acid were different: all-trans was effective at nanomolar concentrations, whereas 10-fold higher levels of 9-cis retinoic acid were required to achieve comparable induction of RAR-beta and CRABP II. Conversely, at high concentrations, 9-cis retinoic acid gave a greater induction of RAR-beta and CRABP II than all-trans. The induction of RAR-beta and CRABP II by all-trans retinoic acid was maintained in the subsequent absence of all-trans retinoic acid, whereas induction by 9-cis retinoic acid was dependent on its continued presence in the culture medium. These results suggest that, at high concentrations, 9-cis retinoic acid may produce its transcriptional effects via retinoid X receptor (RXR) homodimers. This has implications for the cellular functions of 9-cis retinoic acid and its use as a biological response modifier.

Dactinomycin↗

PubMed1996

Induction of the mammalian stress response gene GADD153 by oxidative stress: role of AP-1 element.

GADD153 is a CCAAT/enhancer-binding-protein-related gene that may function to control cellular growth in response to stress signals. In this study, a variety of oxidant treatments were shown to stimulate endogenous GADD153 mRNA expression and to transcriptionally activate a GADD153 promoter-reporter gene construct in transfected HeLa cells. Both commonalities and distinctions in the induction of GADD153 by H2O2 and the thiol-reactive compound arsenite were demonstrated. GADD153 mRNA induction by both H2O2 and arsenite was potentiated by GSH depletion, and completely inhibited by N-acetyl-cysteine. o-Phenanthroline and mannitol blocked GADD153 induction by H2O2, indicating that iron-generated hydroxyl radical mediates this induction. Concordantly, GSH peroxidase overexpression in WI38 cells attenuated GADD153 mRNA induction by H2O2. However, GADD153 induction by arsenite was only modestly reduced in the same cells, suggesting a lesser contribution of peroxides to gene activation by arsenite. We also demonstrated that oxidative stress participates in the induction of GADD153 by UVC (254 nm) irradiation. Finally, both promoter-deletion analysis and point mutation of the AP-1 site in an otherwise intact promoter support a significant role for AP-1 in transcriptional activation of GADD153 by UVC or oxidant treatment. Indeed, exposure of cells to oxidants or UVC stimulated binding of Fos and Jun to the GADD153 AP-1 element. Together, these results demonstrate that both free-radical generation and thiol modification can transcriptionally activate GADD153, and that AP-1 is critical to oxidative regulation of this gene. This study further supports a role for the GADD153 gene product in the cellular response to oxidant injury.

Arsenites↗

PubMed2001

Impaired ultraviolet-B-induced cytokine induction in xeroderma pigmentosum fibroblasts.

Xeroderma pigmentosum is a rare, autosomal recessive disease in which patients develop excessive solar damage at an early age and have a 1000-fold increased risk of developing cutaneous neoplasms. Xeroderma pigmentosum can be classified into seven complementation groups (A-G) with defects in different DNA nucleotide excision repair genes. Xeroderma pigmentosum patients also have impaired immune function including reduced natural killer cell activity and impaired induction of interferon-gamma. We hypothesized that altered cytokine induction may contribute to the immune defect in xeroderma pigmentosum patients. We examined cytokine mRNA expression after ultraviolet B irradiation using reverse transcriptase polymerase chain reaction in fibroblasts derived from five xeroderma pigmentosum patients in complementation groups A, C, and D and in complemented XP-A and XP-D cells. Cytokines interleukin-1beta and interleukin-6 displayed impaired ultraviolet B induction whereas interleukin-8 had normal induction in the xeroderma pigmentosum fibroblasts. Stable complementation of XP-A and XP-D cell lines increased ultraviolet-B-induced interleukin-1beta and interleukin-6 expression. These results demonstrate a deficient response of xeroderma pigmentosum fibroblasts to ultraviolet B in terms of cytokine interleukin-1beta and interleukin-6 induction but normal interleukin-8 induction and exhibit a role for DNA repair in cytokine induction.

Cell Line↗

PubMed2002

The use of anaesthetic rooms for induction of anaesthesia: a postal survey of current practice and attitudes in Great Britain and Northern Ireland.

A postal survey was sent to all anaesthetic departments in the UK to identify current practice and gain insight into anaesthetists' attitudes regarding the use of anaesthetic rooms for induction of general anaesthesia. Replies were received from 247 (88%) departments. Of these, 10 (4%) departments routinely anaesthetise all patients in theatre. The main reason for change was patient safety. Of those who routinely use the anaesthetic room for induction of anaesthesia, only 5% have made provision to change to in-theatre induction. An estimated pound 30 million has been spent on equipping anaesthetic rooms since 1994; with the result that 91% of departments where anaesthetic room induction occurs, now have monitoring that complies with the current Association of Anaesthetists of Great Britain and Ireland guidelines. The majority of the respondents who use anaesthetic rooms perceived induction in theatre to result in reduced efficiency, increased patient anxiety, a worse teaching environment and no improvement in patient safety. This was in contrast to the attitudes of respondents from hospitals where in-theatre induction occurs. Only 9.7% of all respondents believed that clinical governance would necessitate a change to anaesthetizing all patients in theatre compared to 25% who believed that the increasing costs of monitoring equipment would lead to a change. Overall 79% of respondents prefer to use the anaesthetic room, 16% prefer in-theatre induction and 5% expressed no preference. However, of those who routinely anaesthetise in theatre, 70% thought it to be preferable.

Anesthesia Department, Hospital↗

PubMed1999

Pharmacokinetics, induction of anaesthesia and safety characteristics of propofol 6% SAZN vs propofol 1% SAZN and Diprivan-10 after bolus injection.

AIMS: In order to avoid the potential for elevated serum lipid levels as a consequence of long term sedation with propofol, a formulation of propofol 6% in Lipofundin(R) MCT/LCT 10% (Propofol 6% SAZN) has been developed. The pharmacokinetics, induction of anaesthesia and safety characteristics of this new formulation were investigated after bolus injection and were compared with the commercially available product (propofol 1% in Intralipid(R) 10%, Diprivan-10) and propofol 1% in Lipofundin(R) MCT/LCT 10% (Propofol 1% SAZN). METHODS: In a randomised double-blind study, 24 unpremedicated female patients received an induction dose of propofol of 2.5 mg kg-1 over 60 s which was followed by standardized balanced anaesthesia. The patients were randomized to receive propofol as Propofol 6% SAZN, Propofol 1% SAZN or Diprivan-10. RESULTS: For all formulations the pharmacokinetics were adequately described by a tri-exponential equation, as the propofol concentrations collected early after the injection suggested an additional initial more rapid phase. The average values for clearance (CL), volume of distribution at steady-state (Vd,ss ), elimination half-life (t1/2,z ) and distribution half-life (t1/2, lambda2) observed in the three groups were 32+/-1.5 ml kg-1 min-1, 2. 0+/-0.18 l kg-1, 95+/-5.6 min and 3.4+/-0.20 min, respectively (mean+/-s.e.mean, n=24) and no significant differences were noted between the three formulations (P >0.05). The half-life of the additional initial distribution phase (t1/2,lambda1 ) in all subjects ranged from 0.1 to 0.6 min. Anaesthesia was induced successfully and uneventfully in all cases, and the quality of induction was adequate in all 24 patients. The induction time did not vary between the three formulations and the average induction time observed in the three groups was 51+/-1.3 s which corresponded to an induction dose of propofol of 2.1+/-0.06 mg kg-1 (mean+/-s.e. mean, n=24). The percentage of patients reporting any pain on injection did not vary between the formulations and was 17% for the three groups. No postoperative phlebitis or other venous sequelae of the vein used for injection occurred in any of the patients at recovery of anaesthesia nor after 24 h. CONCLUSIONS: From the above results, we conclude that the alteration of the type of emulsion and the higher concentration of propofol in the new parenteral formulation of propofol does not affect the pharmacokinetics and induction characteristics of propofol, compared with the currently available product. Propofol 6% SAZN can be administered safely and has the advantage of a reduction of the load of fat and emulsifier which may be preferable when long term administration of propofol is required.

Adult↗

PubMed2002

Early viral clearance and sustained response in chronic hepatitis C: a controlled trial of interferon and ribavirin after high-dose interferon induction.

High-dose induction with alpha-interferon induces early viral clearance of hepatitis C and combined with ribavirin enhances sustained response. We assess whether adding ribavirin after viral clearance obtained by alpha-interferon induction increased the rate of viral eradication.Forty-one naïve patients with chronic hepatitis C were randomised to receive, after 4 weeks of 10 mU daily of alpha-interferon (induction), 3 mU daily for 22 weeks and 3 mU thrice weekly for 26 weeks of either interferon alone (monotherapy) or interferon plus 1000-1200 mg daily of ribavirin (combination therapy). At the end of the induction phase, 23 (56%) subjects had cleared HCV-RNA. During therapy, breakthrough was observed in four patients on monotherapy, but never in patients on combination therapy. The rate of clearance of HCV-RNA was different between monotherapy and combination therapy at the end of treatment (40% vs. 76.1%, P=0.02) and at the end of follow-up (5% vs. 57.1%, P=0.001). Twelve of the 23 patients who cleared HCV-RNA during induction, but only one of the 18 still HCV-RNA-positive after 4 weeks of therapy, had a sustained response (52.2% vs. 5.6%, P=0.001). Clearance of HCV-RNA at 1 week had a high positive predictive value for sustained response in combination therapy (PPV=0.75), but not in monotherapy (PPV=0.33). Induction with high daily doses of alpha-interferon obtains suppression of hepatitis C in more than half of patients, but ribavirin is needed to maintain a sustained response. The rate of sustained response is a function of the time to HCV-RNA clearance. In patients not responding to induction therapy addition of ribavirin does not obtain a sustained virological response.

Adolescent↗

PubMed2001

Signaling pathways in the induction of c-met receptor expression by its ligand scatter factor/hepatocyte growth factor in human glioblastoma.

Scatter factor/hepatocyte growth factor (SF/HGF) and its tyrosine kinase receptor c-met are developmentally expressed, neuroprotective, and tumorigenic within the CNS. In the present study SF/HGF is shown to induce the expression of c-met in two human glioblastoma cell lines, U-373 MG and T98G, and the signaling pathways involved in this induction are dissected. SF/HGF activated mitogen-activated protein kinase (MAPK) and inhibition of either Ras or MAPK-kinase completely inhibited SF/HGF-mediated c-met induction. Inhibition of phospholipase-C (PLC) did not affect c-met induction in either cell line. Inhibition of phosphoinositide 3-kinase (PI3-kinase) substantially reduced c-met induction by SF/HGF in T98G cells but had no effect in U-373 MG cells. Protein kinase C (PKC) inhibition reduced c-met induction in T98G cells but not in U-373 MG cells. SF/HGF induced the expression of c-fos and c-jun mRNA and increased the levels of AP-1 transcription factor in both cells lines as determined by AP-1-luciferase reporter expression. Transfection of either cell line with TAM-67, a dominant negative for the jun transactivation domain, completely inhibited AP-1 and c-met induction by SF/HGF. These results support a model of c-met induction by SF/HGF in human glioma cells that uniformly involves Ras, MAPK, and AP-1 and additionally involves PI3-kinase and PKC in some cell lines.

Gene Expression Regulation, Neoplastic↗

PubMed2000

alpha-MSH inhibits induction of C/EBPbeta-DNA binding activity and NOS2 gene transcription in macrophages.

BACKGROUND: alpha-Melanocyte-stimulating hormone (alpha-MSH) is an endogenous tridecapeptide that exerts anti-inflammatory actions and abrogates postischemic renal injury in rodents. alpha-MSH inhibits lipopolysaccharide (LPS)-induced gene expression of several cytokines, chemokines, and nitric oxide synthase-2 (NOS2), but the molecular mechanisms underlying these effects have not been clearly defined. To test the hypothesis that alpha-MSH inhibits the expression of inducible trans-activating factors involved in NOS2 regulation, we used RAW 264.7 macrophage cells to examine the effects of alpha-MSH on the activation of nuclear factor-kappaB (NF-kappaB) and CCAAT/enhancer binding protein-beta (C/EBPbeta), trans-acting factors known to be involved in LPS + interferon (IFN)-gamma induction of the NOS2 gene. METHODS: Gel shift assays were performed to identify NF-kappaB and C/EBP DNA binding activities in LPS + IFN-gamma-treated RAW 264.7 cells in the presence and absence of alpha-MSH. NOS2 promoter assays were conducted to identify the effects of alpha-MSH on LPS + IFN-gamma-mediated induction of NOS2 transcription. RESULTS: Gel shift assays demonstrated LPS + IFN-gamma induction of NF-kappaB and C/EBP family protein-DNA complexes in nuclei harvested from the cells. Supershift assays revealed that the C/EBP complexes were comprised of C/EBPbeta, but not C/EBPalpha, C/EBPdelta, or C/EBPepsilon. alpha-MSH (100 nmol/L) inhibited the LPS + IFN-gamma-mediated induction of nuclear DNA binding activity of C/EBPbeta, but not that of NF-kappaB (in contrast to reports in other cell types), as well as the activity of a murine NOS2 promoter-luciferase construct. In contrast, alpha-MSH (100 nmol/L) had no effect on the induction of NOS2 promoter-luciferase genes harboring deletion or mutation of the C/EBP box. CONCLUSIONS: These data indicate that alpha-MSH inhibits the induction of C/EBPbeta DNA binding activity and that this effect is a major mechanism by which alpha-MSH inhibits the transcription of the NOS2 gene. The inability of alpha-MSH to inhibit LPS + IFN-gamma induction of NF-kappaB in murine macrophage cells, which contrasts with inhibitory effects of the neuropeptide in other cell types, suggests that cell-type-specific mechanisms are involved.

Animals↗

PubMed2005

Morbidity and mortality are not increased after induction chemoradiotherapy followed by esophagectomy in patients with esophageal cancer.

Attempts to improve survival of patients with esophageal cancer have been made using induction chemoradiotherapy (CRT) followed by surgery. This approach may be associated with higher complication rates. A large single-center experience was reviewed to determine whether induction CRT was associated with increased morbidity and mortality among 179 patients undergoing esophagectomy between January 1994 and December 2002. Morbidity was recorded as any complication requiring additional intervention. Mortality was defined as patient death within the first 30 postoperative days or death during the initial hospitalization. In total, 131 patients underwent induction CRT followed by surgery. The most common operation was an Ivor-Lewis esophagogastrectomy (60%). Median survival of the entire group was 33 months and 5-year survival was 26%. Perioperative mortality was 5% and did not differ between induction (4.6%) and non-induction (6.3%) groups. The overall complication rate was 57%, reflecting a very liberal definition of postoperative complications. There were no differences between the two groups in terms of such major complications as anastomotic leak, pneumonia, acute respiratory distress syndrome, respiratory failure, chylothorax, atrial arrhythmia, and wound infections. There were only two complications that occurred more frequently in the induction group--venous thrombosis (8.4% v 0%) and vocal cord paresis (7.6% v 2.1%). The median hospital stay was similar in patients who had complications versus those who did not (12 v 13 days) and in patients who underwent induction CRT versus those that did not (12 days v 13.5 days). Esophagectomy can be performed safely with low mortality and acceptable morbidity following neoadjuvant chemotherapy and radiation with no increase in hospital stay for patients with complications.

Adenocarcinoma↗

PubMed1992

Induction of labor as compared with serial antenatal monitoring in post-term pregnancy. A randomized controlled trial. The Canadian Multicenter Post-term Pregnancy Trial Group.

BACKGROUND: The rates of perinatal mortality and neonatal morbidity are higher for post-term pregnancies than for term pregnancies. It is not known, however, whether the induction of labor results in better outcomes than does serial fetal monitoring while awaiting spontaneous labor. METHODS: We studied 3407 women with uncomplicated pregnancies of 41 or more weeks' duration. The women were randomly assigned to undergo induction of labor or to have serial antenatal monitoring and spontaneous labor unless there was evidence of fetal or maternal compromise, in which case labor was induced or cesarean section was performed. In the induction group, labor was induced by the intracervical application of prostaglandin E2. Serial antenatal monitoring consisted of counts of fetal kicks, nonstress tests, and assessments of amniotic-fluid volume. The outcomes we measured were the rates of perinatal mortality, neonatal morbidity, and delivery by cesarean section. RESULTS: Among the 1701 women in the induction group, 360 (21.2 percent) underwent cesarean section, as compared with 418 (24.5 percent) of the 1706 women in the monitoring group (P = 0.03). This difference resulted from a lower rate of cesarean section performed because of fetal distress among the women in the induction group (5.7 percent vs. 8.3 percent, P = 0.003). When two infants with lethal congenital anomalies were excluded, there were no perinatal deaths in the induction group and two stillbirths in the monitoring group (P not significant). The frequency of neonatal morbidity was similar in the two groups. CONCLUSIONS: In post-term pregnancy, the induction of labor results in a lower rate of cesarean section than serial antenatal monitoring; the rates of perinatal mortality and neonatal morbidity are similar with the two approaches to management.

Adult↗

PubMed2003

Efficacy and safety of tracheobronchoplasty after induction therapy for locally advanced lung cancer.

OBJECTIVES: Patients receiving induction therapy may have increased risk of morbidity and mortality after surgery. We retrospectively evaluated the influence of preoperative treatment in patients who underwent sleeve resection for lung cancer. METHODS: A series of 48 consecutive patients who underwent sleeve resection with a telescoping anastomosis was retrospectively analyzed. A sleeve lobectomy and pneumonectomy were performed in 41 and 7 patients, respectively. Twenty patients received preoperative induction therapy; of them, 16 received induction chemoradiotherapy and 4 received only chemotherapy. Twenty-eight patients underwent the procedure without adjuvant therapy. RESULTS: The telescopic procedure was performed by placing sutures around the proximal and distal portions of the bronchial cartilage without wrapping the anastomosis. Among the 20 patients who received induction therapy, pulmonary angioplasty was performed in 5 and chest wall resection was performed in 3. Seven of these 20 patients (35%) had postoperative complications. Among the 28 patients without preoperative adjuvant therapy, pulmonary angioplasty was performed in 3, diaphragmatic resection was performed in 1, and chest wall resection was performed in 1. Three of these 28 patients (11%) had postoperative complications. Complications relating to the anastomosis occurred in 1 patient (5.0%) who received induction therapy; however, no operative deaths occurred. Bronchoscopic examinations demonstrated that mucosal healing was prolonged in patients who underwent induction therapy. CONCLUSION: Induction therapy did not significantly affect morbidity or mortality among patients who underwent sleeve resection.

Adenocarcinoma↗

PubMed1976

Induction and decay of glucagon-induced amino acid transport in primary cultures of adult rat liver cells: paradoxical effects of cycloheximide and puromycin.

Liver parenchymal cells were isolated from adult rats and cultured in collagen-coated plastic petri dishes in serum-free medium. Glucagon induced 4- to 5-fold increases in alpha-aminoisobutyric acid (AIB) transport within 6 hr. Dexaemthasone had no direct effect on AIB transport but greatly potentiated the induction by glucagon ("permissive effect"). Levels of 3':5'-cyclic AMP increased 30- to 100-fold within 30 min after glucagon addition to cultures that had been treated with dexamethasone, and dibutyryl cyclic AMP mimicked the glucagon induction of AIB transport. Additionally, dexamethasone exerted a "permissive" effect on induction of AIB transport by dibutyryl cyclic AMP, whereas the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine induced AIB transport only in cultures that had been treated with dexamethasone. Induction of AIB transport was not dependent upon the continued presence of glucagon, but induced AIB transport activity decayed to uninduced levels within 3-4 hr after glucagon removal. The protein syntesis inhibitors puromycin and cycloheximide inhibited both induction and decay of glucagon induced AIB transport, but had a stabilizing effect if added once induction or decay had commenced. Unlike cycloheximide, the inhibitory effect of puromycin on the glucagon induction of AIB transport was reversible.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed1991

Widespread expression of MyoD genes in Xenopus embryos is amplified in presumptive muscle as a delayed response to mesoderm induction.

The MyoD gene codes for an important regulatory factor in skeletal myogenesis. To explore the relationship between mesoderm induction in Xenopus embryos and expression of MyoD, I have monitored MyoD mRNA levels in normal embryos and cultured explants by RNase protection. Transcription from the two Xenopus MyoD gene copies is activated weakly across the whole embryo at the midblastula transition, and this activation occurs in the absence of mesoderm induction. In response to induction this basal expression is amplified 50- to 100-fold, but in animal-pole explants 6-10 hr elapse before induced mRNAs appear, and this induction requires prior protein synthesis. The promiscuous transcripts disappear from animal explants at a time when induction "competence" is lost, suggesting a link between these events. The data highlight a broad, but transient, permissiveness for MyoD expression in embryos, which is propagated and amplified only in presumptive muscle in response to induction. Moreover, muscle-specific MyoD expression is a relatively late (postgastrulation) event in the mesoderm-induction cascade.

Actins↗

PubMed1999

Collaborative role of E2F transcriptional activity and G1 cyclindependent kinase activity in the induction of S phase.

A considerable body of evidence points to a role for both cyclin E/cyclin-dependent kinase (cdk)2 activity and E2F transcription activity in the induction of S phase. We show that overexpression of cyclin E/cdk2 in quiescent cells induces S phase, that this coincides with an induction of E2F activity, and that coexpression of E2F enhances the cyclin E/cdk2-mediated induction of S phase. Likewise, E2F overexpression can induce S phase and does so in the apparent absence of cyclin E/cdk2 activity. In addition, although the inhibition of cyclin E/cdk2 activity blocks the induction of S phase after growth stimulation of normal mouse embryo fibroblasts, inhibition of cyclin E/cdk2 does not block S phase induction in Rb-/- cells where E2F activity is deregulated. These results point to the important roles for E2F and cyclin E/cdk2 in the induction of S phase. Moreover, the nature of the E2F targets and the suspected targets for cyclin E/cdk2 suggests a potential molecular mechanism for the collaborative action of cyclin E/cdk2 and E2F in the induction of S phase.

Animals↗

PubMed1995

Mechanism of the tumor necrosis factor alpha-mediated induction of endothelial tissue factor.

This study examines the regulation of the human tissue factor (TF) promotor in vitro and in vivo. Transient transfections were performed in bovine aortic endothelial cells to investigate the role of two fundamentally different AP-1 sites and a closely located NF-kappa B site in the human TF promoter. The NF-kappa B site is functionally active, since overexpression of NF-kappa B(p65) resulted in induction of TF mRNA and activity. Promoter analysis showed that NF-kappa B induction was dependent on the integrity of the region from base pair -188 to -181. Over-expression of Jun/Fos resulted in TF induction of transcription and protein/activity. Functional studies revealed that the proximal AP-1 site, but not the distal, was inducible by Jun/Fos heterodimers. The distal AP-1 site, which has a G-->A switch at position 4, was inductible by Jun homodimers. Electrophoretic mobility shift assays, using extracts of tumor necrosis factor alpha (TNF alpha)-stimulated bovine aortic endothelial cells, demonstrated TNF alpha-inducible binding to the proximal AP-1 site, comprising JunD/Fos heterodimers. At the distal AP-1 site, only minor induction of binding activity, characterized as proteins of the Jun and ATF family, was observed. Consistently, this site only marginally participates in TNF alpha induction. Functional studies with TF promotor plasmids confirmed that deletion of the proximal AP-1 or the NF-kappa B site decreased TNF alpha-mediated TF induction to a higher extend than loss of the distal AP-1 site. However, integrity of both AP-1 sites and the NF-kappa B site was required for optimal TNF alpha stimulation. The relevance of these in vitro data was confirmed in vivo in a mouse tumor model. Expression plasmids for a dominant negative Jun mutant or I-kappa B were packaged in liposomes. When either mutated Jun or I-kappa B were injected intravenously 48 h before TNF alpha, a reduction in TNF alpha-mediated TF expression in the tumor endothelial cells was observed. Simultaneously, fibrin/fibrinogen deposition decreased and free blood flow could be restored. Thus, TNF alpha-induced up-regulation of endothelial cell TF depends on a concerted action of members of the bZIP and NF-kappa B family.

Animals↗

PubMed1996

Induction of p21 mediated by reactive oxygen species formed during the metabolism of aziridinylbenzoquinones by HCT116 cells.

Aziridinylbenzoquinones are a group of antitumor agents that elicit cytotoxicity by generating either alkylating intermediates or reactive oxygen species. The mechanism of toxicity may not always, however, involve profound damage of cellular constituents, but may involve a cytostatic effect through interference with the cell cycle. In this context, we have examined the induction of the cell cycle inhibitor p21 (WAF1, CIP1, or sdi1), whose overexpression suppresses the growth of various tumor cells, in human tumor cells metabolizing 3,6-diaziridinyl-1,4-benzoquinone (DZQ) and its C2,C5-substituted derivatives: 2,5-bis-(carboethoxyamino) (AZQ) and 2, 5-bis-2(-hydroxyethylamino) (BZQ). Both DZQ and AZQ were effectively activated by HCT116 human colonic carcinoma cells; the activation of the former involved largely a dicoumarol-sensitive activity, whereas that of the latter appeared to be accomplished primarily by one-electron transfer reductases. BZQ was not a substrate for the dicoumarol-sensitive enzyme in HCT116 cells. Cellular activation of the first two quinones was associated with formation of oxygen-centered radicals as detected by EPR in conjunction with the spin trap 5,5'-dimethyl-1-pyrroline-N-oxide. The redox transitions of DZQ involved hydroxyl radical formation and were strongly inhibited by catalase, whereas those of AZQ showed a strong superoxide anion component sensitive to superoxide dismutase. These signals were suppressed by N-acetylcysteine with concomitant production of a thiyl radical adduct. This suggests an effective electron transfer between the thiol and free radicals formed during the activation of these quinones. DZQ and AZQ induced significantly the expression of p21 in HCT116 cells, but a 10-fold higher concentration of AZQ was required to achieve the level of induction elicited by DZQ. BZQ had little effect on p21 expression. p21 induction at both mRNA and protein levels correlated with the inhibition of either cyclin-dependent kinase activity or cell proliferation. p21 induction elicited by the above quinones was inhibited by N-acetylcysteine, whereas the non-sulfur analog, N-acetylalanine, was without effect. Catalase and superoxide dismutase did not effect p21 induction by aziridinylbenzoquinones in HCT116 cells, thus suggesting that extracellular sources of oxygen radicals generated by plasma membrane reductases have no influence in the expression of this gene. Hydrogen peroxide, a product of quinone redox cycling, elicited an increase of p21 mRNA levels in HCT116 and K562 human chronic myelogenous leukemia cells. The latter lacks p53, one of the activators of p21 transcription, thus suggesting that p21 expression can be accomplished in a p53-independent manner in these cells. This study suggests that p21 induction is mediated by an increase in the cellular steady-state concentration of oxygen radicals and that the greater effectiveness in p21 induction by DZQ may be related to its efficient metabolism by NAD(P)H:quinone oxidoreductase activity in HCT116 cells.

Acetylcysteine↗

PubMed1998

p38 kinase activity is essential for osmotic induction of mRNAs for HSP70 and transporter for organic solute betaine in Madin-Darby canine kidney cells.

In renal cells, hypertonicity induces genes for heat shock proteins (HSP70, alpha B-crystallin), as well as enzymes and transporters directly involved in the metabolism and transport of protective organic osmolytes. While heat shock proteins are induced by many stresses including osmotic stress, the induction of the osmolytes genes appears to be specific to osmotic stress. These two adaptive mechanisms allow kidney cells to survive and function in the hypertonic environment that exists on routine basis in kidney medulla. In mammalian cells, hypertonicity induces three mitogen-activated protein kinase pathways: ERK (extracellular regulated kinase), JNK (Jun N-terminal kinase), and p38. ERK activation by osmotic stress is a consistent finding in many cells, but it is not essential for transcriptional regulation of mRNA for transporter of organic osmolyte betaine. While the growth of yeast cells on NaCl-supplemented medium is dependent on HOG1 pathway, it is still unclear which pathway mediates the adaptation to osmotic stress in mammalian cells. Here, we show that inhibition of p38 kinase activity, using the specific inhibitor SB203580 (4-(fluorophenyl)-2-(4-methylsulfonyl-phenyl)-5-(4-pyridyl) imidazole), abolishes the hypertonicity-mediated induction of mRNAs for HSP70 and betaine transporter in Madin-Darby canine kidney cells. The inhibition is dose-dependent and correlates with the in situ activity of native p38 kinase, determined as MAPKAPK-2 activity in cell extracts. As reported previously, the activities of ERK-1 and -2 were not affected by SB203580, but surprisingly, inhibition of native p38 kinase activity correlates with up-regulation of native JNK-1 activity in osmotically stressed cells. p38 mRNA is induced by hypertonic stress and is attenuated with p38 kinase inhibition. We also find that thermal induction of HSP70 mRNA is not affected by p38 kinase inhibition. Such findings suggest that p38 kinase activity is essential for the induction of genes involved in the adaptation of mammalian cells to osmotic stress and that the increased activity of JNK-1 during p38 kinase inhibition is consistent with regulation of JNK-1 by p38 kinase in osmotically stressed cells. In addition, the transduction pathways mediating HSP70 mRNA induction by different stresses appear to be divergent; osmotic induction of HSP70 is p38 kinase-dependent, while thermal induction is not.

Animals↗