Merbarone, a catalytic inhibitor of DNA topoisomerase II, induces apoptosis in CEM cells through activation of ICE/CED-3-like protease.
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Merbarone (5-[N-phenyl carboxamido]-2-thiobarbituric acid) is an anticancer drug that inhibits the catalytic activity of DNA topoisomerase II (topo II) without damaging DNA or stabilizing DNA-topo II cleavable complexes. Although the cytotoxicity of the complex-stabilizing DNA-topo II inhibitors such as VP-16 (etoposide) has been partially elucidated, the cytotoxicity of merbarone is poorly understood. Here, we report that merbarone induces programmed cell death or apoptosis in human leukemic CEM cells, characterized by internucleosomal DNA cleavage and nuclear condensation. Treatment of CEM cells with apoptosis-inducing concentrations of merbarone caused activation of c-Jun NH2-terminal kinase/stress-activated protein kinase, c-jun gene induction, activation of caspase-3/CPP32-like protease but not caspase-1, and the proteolytic cleavage of poly(ADP-ribose) polymerase. Treatment of CEM cells with a potent inhibitor of caspases, Z-Asp-2. 6-dichlorobenzoyloxymethyl-ketone, inhibited merbarone-induced caspase-3/CPP32-like activity and apoptosis in a dose-dependent manner. These results indicate that the catalytic inhibition of topo II by merbarone leads to apoptotic cell death through a caspase-3-like protease-dependent mechanism. These results further suggest that c-Jun and c-Jun NH2-terminal kinase/stress-activated protein kinase signaling may be involved in the cytotoxicity of merbarone. (+info)
Selective loss of PMA-stimulated expression of matrix metalloproteinase 1 in HaCaT keratinocytes is correlated with the inability to induce mitogen-activated protein family kinases.
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Many cell types, including fibroblasts and primary keratinocytes, increase matrix metalloproteinase 1 (MMP-1) production in response to agonists such as growth factors and phorbol esters. However, the spontaneously transformed human keratinocyte cell line HaCaT, although it increases MMP-1 production in response to epidermal growth factor (EGF), does not respond similarly to stimulation with PMA. This phenomenon occurs even though HaCaT cells remain proliferatively responsive to both agonists, suggesting a HaCaT-specific defect in a PMA-mediated signal transduction pathway. Using an inside-out approach to elucidate the source of this defect, we found that EGF, but not PMA, stimulated MMP-1 promoter activity in transiently transfected HaCaT keratinocytes. In addition, an assessment of fibroblast and HaCaT c-fos and c-jun gene expression after exposure to EGF and PMA showed that although both agonists increased the expression of c-fos and c-jun mRNA in fibroblasts, only EGF did so in HaCaT keratinocytes. Finally, we looked at the activation of mitogen-activated protein (MAP) family kinases after stimulation with EGF or PMA and found that both agonists increased the phosphorylation and activation of fibroblast extracellular signal-regulated protein kinase and c-Jun N-terminal kinase, but only EGF activated the same kinase activities in HaCaT cells. Further, the EGF-mediated increase in MMP-1 gene expression was inhibited by the MAP kinase/ERK kinase (MEK)-specific inhibitor PD98059 and the p38 kinase-specific inhibitor SB203580. Our evidence indicates that although HaCaT MAP kinases are functional, they are not properly regulated in response to the activation of protein kinase C, and that the defect that bars HaCaT MMP-1 expression in response to stimulation with PMA lies before MAP kinase activation. (+info)
Immediate-early gene expression in the inferior mesenteric ganglion and colonic myenteric plexus of the guinea pig.
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Activation of neurons in the inferior mesenteric ganglion (IMG) was assessed using c-fos, JunB, and c-Jun expression in the guinea pig IMG and colonic myenteric plexus during mechanosensory stimulation and acute colitis in normal and capsaicin-treated animals. Intracolonic saline or 2% acetic acid was administered, and mechanosensory stimulation was performed by passage of a small (0.5 cm) balloon either 4 or 24 hr later. Lower doses of capsaicin or vehicle were used to activate primary afferent fibers during balloon passage. c-Jun did not respond to any of the stimuli in the study. c-fos and JunB were absent from the IMG and myenteric plexus of untreated and saline-treated animals. Acetic acid induced acute colitis by 4 hr, which persisted for 24 hr, but c-fos was found only in enteric glia in the myenteric plexus and was absent from the IMG. Balloon passage induced c-fos and JunB in only a small subset of IMG neurons and no myenteric neurons. However, balloon passage induced c-fos and JunB in IMG neurons (notably those containing somatostatin) and the myenteric plexus of acetic acid-treated animals. After capsaicin treatment, c-fos and JunB induction by balloon passage was inhibited in the IMG, but there was enhanced c-fos expression in the myenteric plexus. c-fos and JunB induction by balloon stimulation was also mimicked by acute activation of capsaicin-sensitive nerves. These data suggest that colitis enhances reflex activity of the IMG by a mechanism that involves activation of both primary afferent fibers and the myenteric plexus. (+info)
Rat embryo fibroblasts transformed by c-Jun display highly metastatic and angiogenic activities in vivo and deregulate gene expression of both angiogenic and antiangiogenic factors.
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The comparative tumorigenicity in rats and nude mice of cell lines derived from FR3T3 and transformed by either c-jun, ras, SV40 lt, or bovine papilloma virus type 1 (BPV1) oncogenes was investigated. c-Jun-transformed cells were as tumorigenic and metastatic as Ras-transformed cells. Latencies were short, and numerous pulmonary metastases were observed in all injected animals. In contrast, tumors induced by s.c. injection of SV40-transformed cells developed slower, and none of the animals who received injections i.v. presented with metastases. BPV1-transformed cells had an intermediate tumorigenic and metastatic activity. Microvessels present in the different tumors were revealed by immunostaining with Griffonia (Bandeiraea) Simplicifolia lectin 1. Tumors obtained with c-Jun-transformed cells exhibited more neovascularization than those induced by the other oncogenes. By comparison to FR3T3 cells or SV40- or BPV1-transformed cells, c-Jun-transformed fibroblasts repress the antiangiogenic thrombospondin-1 and SPARC genes, whereas we found that they express higher levels of gene expression of the angiogenic vascular endothelial growth factor. Finally, as compared with cells before passage in animals, thrombospondin-1, SPARC, and VEGF gene expression was also deregulated in cell lines isolated from primary tumors induced by BPV1-transformants. Our results indicate that the high transforming potential of c-Jun, evidenced as soon as transformation is established in vitro, correlates with deregulation of gene expression of both angiogenic and antiangiogenic factors leading to rapid neovascularization of tumors. (+info)
Engagement of natural cytotoxicity programs regulates AP-1 expression in the NKL human NK cell line.
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NK cell cytotoxicity is a fast and efficient mechanism of target cell lysis. Using transcription analysis, such as multiplex messenger assays, we show here that natural cytotoxicity exerted by the human NKL cell line correlates with mRNA accumulation of very early activator protein (AP)-1 transcription factor genes such as JunB, FosB and c-Fos. In addition, DNA-binding activities of Jun-Fos heterodimers were observed by electrophoretic mobility shift assays during the course of natural cytotoxicity. Interaction between immunoglobulin-like transcript-2/leukocyte Ig-like receptor 1 on NKL cells and HLA-B27 on target cells leads to an impairment of NKL natural cytotoxicity, which correlates with an absence of JunB, FosB, and c-Fos transcription, as well as an absence of their DNA-binding activity. Our studies thus indicate that, despite the rapidity of NK cell-mediated lysis, AP-1 transcription factor is activated during the early stage of NK cell cytolytic programs and that engagement of NK cell inhibitory receptors for MHC class I molecules impairs the very early activation of AP-1. (+info)
Human cytomegalovirus IE1 protein activates AP-1 through a cellular protein kinase(s).
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During human cytomegalovirus (HCMV) infection, a rapid increase in AP-1 activity is detected. In this study, activation of transcription from promoters containing AP-1-binding sites by the IE1 protein of HCMV was examined. In transient transfection assays with reporter plasmids, it was found that IE1 strongly induced AP-1-driven transcription. Cells stably expressing IE1 also showed higher levels of AP-1 activity than did control cells. IE1 expression did not raise levels of c-jun and c-fos RNA, as determined by quantitative RT-PCR. AP-1 induction by IE1 was blocked efficiently by protein kinase inhibitors in a cell type-dependent manner; for example, by staurosporine in the human microglial cell line U373MG and by H7 in the human promonocytic cell line U937. IE1-driven activation of AP-1 was increased dramatically by mitogen-activated protein kinase/extracellular signal-regulated kinase kinase kinase 1 (MEKK1). The results of this study indicate that IE1 activates AP-1 at the post-transcriptional level and that MEKK1 may play an important role in this process. (+info)
Anchorage dependence of mitogen-induced G1 to S transition in primary T lymphocytes.
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Anchorage dependence defines the cellular requirement for integrin-mediated adhesion to substrate to initiate DNA replication in response to growth factors. In this study we investigated whether normal T cells, which spend extended periods in a nonadherent state, show similar requirements for cell cycle progression in response to TCR stimulation. Resting primary T lymphocytes were induced to enter the cell cycle by TCR triggering, and leukocyte integrins were either engaged using purified ICAM-1 or inhibited with function-blocking mAbs. Our data indicate that leukocyte integrins complement TCR-driven mitogenic signals not as a result of their direct clustering but, rather, via integrin-dependent organization of the actin cytoskeleton. Leukocyte integrin-dependent reorganization of the actin cytoskeleton cooperates with the TCR to effect mitogen-activated protein kinase activation, but also represents a required late (4-8 h poststimulation) component in the mitogenic response of normal T cells. Prolonged leukocyte integrin-dependent spreading, in the context of intercellular contact, is a requisite for the production of the mitogenic cytokine IL-2, which, in turn, is involved in the induction of D3 cyclin and is primarily responsible for the decrease in the cyclin-dependent kinase inhibitor p27kip, resulting in retinoblastoma protein inactivation and S phase entry. Thus, T lymphocytes represent a peculiar case of anchorage dependence, in which signals conveyed by integrins act sequentially with the activating stimulus to effect a sustained production of the essential mitogenic cytokine. (+info)
The N-terminal transactivation domain of ATF2 is a target for the co-operative activation of the c-jun promoter by p300 and 12S E1A.
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The adenovirus E1A proteins activate the c-jun promoter through two Jun/ATF-binding sites, jun1 and jun2. P300, a transcriptional coactivator of several AP1 and ATF transcription factors has been postulated to play a role in this activation. Here, we present evidence that p300 can control c-jun transcription by acting as a cofactor for ATF2: (1) Over-expression of p300 was found to stimulate c-jun transcription both in the presence and absence of E1A. (2) Like E1A, p300 activates the c-jun promoter through the junl and jun2 elements and preferentially activates the N-terminal domain of ATF2. (3) Co-immunoprecipitation assays of crude cell extracts indicate that endogenous p300/CBP(-like) proteins and ATF2 proteins are present in a multiprotein complex that can bind specifically to the jun2 element. We further demonstrate that the Stress-Activated-Protein-Kinase (SAPK) target sites of ATF2, Thr69 and Thr71 are not required for the formation of the p300/CBP-ATF2 multiprotein complex. These data indicate that E1A does not inhibit all transcription activation functions of p300, and, in fact, cooperates with p300 in the activation of the ATF2 N-terminus. (+info)