Studies of the Relationship Between Cell Proliferation and Cell Death. III. AP-1 DNA-Binding Activity during Concanavalin A-Induced Proliferation or Dexamethasone-Induced Apoptosis of Rat Thymocytes

1993 ◽  
Vol 192 (2) ◽  
pp. 386-391 ◽  
Author(s):  
E. Sikora ◽  
E. Grassilli ◽  
E. Bellesia ◽  
L. Troiano ◽  
C. Franceschi
Keyword(s):  
Cell Proliferation ◽  
Cell Death ◽  
Dna Binding ◽  
Concanavalin A ◽  
Binding Activity ◽  
Dna Binding Activity ◽  
Induced Apoptosis ◽  
The Relationship

scholarly journals p53 regulates cyclophosphamide teratogenesis by controlling caspases 3, 8, 9 activation and NF-κB DNA binding

Reproduction ◽  
2007 ◽  
Vol 134 (2) ◽  
pp. 379-388 ◽  
Author(s):  
Olga Pekar ◽  
Nataly Molotski ◽  
Shoshana Savion ◽  
Amos Fein ◽  
Vladimir Toder ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Dna Binding ◽  
Molecular Mechanisms ◽  
Binding Activity ◽  
Tumor Suppressor Protein ◽  
Dna Binding Activity ◽  
Organ Level ◽  
Induced Apoptosis ◽  
The Brain ◽  
Transcription Factor Nuclear Factor

The tumor suppressor protein p53 regulates the sensitivity of embryos to such human teratogens as ionizing radiation, diabetes, and cytostatics. Yet, the molecular mechanisms whereby it fulfills this function remain undefined. We used p53 heterozygous (p53+/−) female mice mated with p53+/− males and then exposed to cyclophosphamide (CP) to test whether caspases 3, 8, and 9 and the transcription factor nuclear factor (NF)-κB may serve as p53 targets. Mice were exposed to CP on day 12 of pregnancy and killed on days 15 and 18 of pregnancy to evaluate CP-induced teratogenic effect. The brain and limbs of embryos harvested 24 h after CP treatment were used to evaluate NF-κB (p65) DNA-binding activity by an ELISA-based method, the activity of the caspases by appropriate colorimetric kits, apoptosis, and cell proliferation by TUNEL, and 5′-bromo-2′-deoxyuridine incorporation respectively. We observed that the activation of caspases 3, 8, and 9 and the suppression of NF-κB DNA binding following CP-induced teratogenic insult took place only in teratologically sensitive organs of p53+/+ but not p53−/− embryos. CP-induced apoptosis and suppression of cell proliferation were also more intensive in the former, and they exhibited a higher incidence of structural anomalies, such as open eyes, digit, limb, and tail anomalies. The analysis of the correlations between the p53 embryonic genotype, the activity of the tested molecules, and the CP-induced dysmorphic events at the cellular and organ level suggests caspases 3, 8, and 9 and NF-κB as components of p53-targeting mechanisms in embryos exposed to the teratogen.

scholarly journals Suppression of E1A-Mediated Transformation by the p50E4F Transcription Factor

1999 ◽  
Vol 19 (7) ◽  
pp. 4739-4749 ◽  
Author(s):  
Elma R. Fernandes ◽  
Robert J. Rooney
Keyword(s):  
Cell Death ◽  
Transcription Factor ◽  
Suppressive Effect ◽  
Culture Conditions ◽  
Binding Activity ◽  
Cellular Transcription Factor ◽  
Dna Binding Activity ◽  
3T3 Fibroblasts ◽  
Induced Apoptosis ◽  
Nih 3T3

ABSTRACT The adenovirus E1A gene can act as an oncogene or a tumor suppressor, with the latter effect generally arising from the induction of apoptosis or the repression of genes that provide oncogenic growth stimuli (e.g., HER-2/c-erbB2/neu) or increased metastatic invasiveness (e.g., metalloproteases). In this study, coexpression of E1A and p50E4F, a cellular transcription factor whose DNA binding activity is stimulated by E1A, suppressed colony formation by NIH 3T3 cells and transformation of primary rat embryo fibroblasts but had no observed effect in the absence of E1A. Domains in p50E4F required for stimulation of the adenovirus E4 promoter were required for the suppressive effect, indicating a transcriptional mechanism. In serum-containing media, retroviral expression of p50E4F in E1A13S/ras-transformed NIH 3T3 fibroblasts had little effect on subconfluent cultures but accelerated a decline in viability after the cultures reached confluence. Cell death occurred by both apoptosis and necrosis, with the predominance of each process determined by culture conditions. In serum-free media, p50E4F accelerated E1A-induced apoptosis. The results suggest that p50E4F sensitizes cells to signals or conditions that cause cell death.

Molecular Mechanisms of Parthenolide-Mediated Pro-Apoptotic Activity in Acute Myeloid Leukemia Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2463-2463
Author(s):  
Mohammad Minhajuddin ◽  
Shanshan Pei ◽  
John M Ashton ◽  
Kevin Callahan ◽  
Eleni Lagadinou ◽  
...  
Keyword(s):  
Cell Death ◽  
Transcription Factor ◽  
Dna Binding ◽  
Myeloid Leukemia ◽  
Molecular Mechanisms ◽  
Lentiviral Vector ◽  
Binding Activity ◽  
Dna Binding Activity ◽  
Apoptotic Activity

Abstract Abstract 2463 Acute myeloid leukemia is malignant disease, characterized by an accumulation of immature myeloid cells. Recent studies have demonstrated that myeloid leukemia appears to arise from a population of leukemia stem cells (LSCs). LSCs typically reside in a quiescent state and therefore do not respond to standard chemotherapeutic agents, which generally target more actively dividing cells. However, LSCs do display certain unique molecular properties that can be exploited to target this relatively rare population of cells that drive disease pathogenesis. Specifically, NF-kB, a pro-survival transcription factor, is constitutively active in LSCs but not in normal hematopoietic stem cells (HSCs). Targeting this pathway by pharmaceutical approaches has been suggested as a potential strategy in the treatment of leukemia; however, inhibiting this pathway alone is not sufficient to strongly induce AML-specific cell death. Further investigation of pathways, that are unique to AML, is a key in designing more effective pharmacologic agents that specifically target the LSC. We have previously demonstrated that the naturally occurring compound parthenolide (PTL) induces apoptosis in primary AML cells, including the stem and progenitor cell. While the empirical anti-leukemic activity of PTL is clear, the underlying molecular mechanisms remain poorly understood. Here we investigate two properties associated with parthenolide-mediated cell death: i) activation of pro-apoptotic transcription factor p53, ii) inhibition of pro-survival transcription factor NF-kB. In order to evaluate the role of p53 signaling, AML cells were challenged with PTL resulting in the phosphorylation of p53 at serine-15, indicating activation p53 in response to PTL. To further investigate the role of p53 in PTL mediated responses, we generated a lentiviral vector expressing shRNAs specifically targeting p53. Leukemia cells were infected with the lentiviral vector encoding p53 shRNA or scramble control and evaluated by qPCR and western blot analysis. The data showed a significant knockdown of p53 mRNA and protein levels, as well as strong inhibition of p21 expression, indicating the specificity of p53 knockdown. Exposure of cells to PTL in which p53 has been specifically disrupted results in partial rescue from PTL mediated cell death, implicating the role of p53 in this response. Next, we performed a detailed analysis of the molecular mechanism by which PTL inhibits NF-kB pathway activity. Using a novel analog of PTL, we demonstrate that the compound directly binds to IKK-beta. Upon exposure to PTL, IKK-beta shows reduced kinase activity, indicating that binding of the drug directly impairs enzymatic function. Secondary to the inhibition of IKK-beta kinase activity, there is decreased phosphorylation of IkB-alpha at ser32/36, resulting in reduced proteosome mediated degradation. As expected, translocation of RelA/p65 to the nucleus was also impaired, resulting in decreased DNA binding activity as evidenced by electrophoretic mobility shift assay (EMSA). Interestingly, studies with a biotinylated analog also show that PTL appears to directly bind p65, we also observed a decreased phosphorylation of p65 at serine 536, an event mediating the transcriptional activity of DNA-bound p65. Inhibition of the NF-kB pathway by parthenolide also resulted in very significant inhibition of one of its well-known downstream target genes, ICAM-1 (CD54) at mRNA, protein and surface expression levels. Whether reduced ICAM-1 expression affects the biology of AML cells is as yet unknown. However, given the known role of ICAM-1 in integrin signaling, we propose that loss of ICAM-1 via NF-kB inhibition may impair the ability of AML cells to interact with their environment. Taken together, this study further elucidates the mechanisms by which PTL mediates pro-apoptotic activity in primary AML cells. PTL induces activation of p53 pathway and therefore knockdown of p53 by shRNA results in partial rescue from PTL mediated cell death. PTL also inhibits the NF-kB pathway, which includes binding of PTL to both IKK-beta and RelA/p65, which leads to decreased phosphorylation of IkB-alpha and reduced DNA binding of p65. In addition, we have discovered the ICAM-1 expression in AML cells is regulated by NF-kB, and that loss of NF-kB DNA binding activity results in loss of ICAM-1 expression. Disclosures: No relevant conflicts of interest to declare.

The Microenvironmental Stromal Cells Abrogate NF-κb Inhibitor Induced Apoptosis in Chronic Lymphocytic Leumkemia

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3208-3208
Author(s):  
Katharina Foerster ◽  
Carl Philipp Simon-Gabriel ◽  
Dorothee Bleckmann ◽  
Marco Benkisser-Petersen ◽  
Nicolas Thornton ◽  
...  
Keyword(s):  
Dna Binding ◽  
Cell Viability ◽  
Cultured Cells ◽  
Cd40 Ligand ◽  
Binding Activity ◽  
Parp Cleavage ◽  
Dna Binding Activity ◽  
Induced Apoptosis

Abstract Introduction: In recent years, the emergence of kinase inhibitors has drastically altered treatment strategies and improved outcomes in CLL patients, but lack of cure and resistance to therapy still remain serious issues. The transcription factor NF-κB influences several cellular functions such as proliferation, apoptosis and inflammation and is known as a key factor contributing to CLL development and progression. NF-κB is constitutively active in CLL and the NF-κB subunit RELA has been proposed as a prognostic marker in CLL with high RELA DNA-binding activity being predictive of short time to first treatment and overall survival. Therefore, NF-κB has gained attention as a promising therapeutic target. NF-kB inhibition induces apoptosis in CLL cells in vitro. However, whether this effect pertains in vivoremains unclear. Since the microenvironment is crucial for CLL cell viability circumventing apoptosis, we tested whether NF-κB inhibition modulates CLL viability in the presence of the microenvironment. Methods: The specific NF-κB inhibitor Dehydroxymethylepoxyquinomicin (DHMEQ) was used alone (2-5 µg/ml) or combined with fludarabine (10 µM), rhBAFF (50 ng/ml), rhAPRIL (500 ng/ml), rhSDF-1a (100 ng/ml) or CD40 ligand (1 µg/ml) on primary CLL cells cultured alone (monoculture) or on bone marrow stromal cells (BMSC) (co-culture with a ratio of 20 CLL cells per stromal cell) for 48-144 h. Viability and apoptosis were measured by flow cytometry using AnnexinV/PI stainings. Protein expression was analyzed by western blot using standard protocols. NF-κB DNA-binding activity after DHMEQ treatment (5 µg/ml) for 6 h was measured by ELISA for all subunits using 1 µg of protein lysate for the NF-κB1 subunit and 10 µg protein lysate for the subunits RELA, NF-κB2, RELB and c-REL. RELA gene knockdown was performed by siRNA transfection (2 µM targeting and non-targeting siRNA). Results: NF-κB inhibition using DHMEQ led to apoptosis in monocultured CLL cells (viability 74% vs. 24%, n=17, p<0.0001) but surprisingly had no effect on cell viability of cells co-cultured with BMSC (viability 96% vs. 95%, p=0.9995). In monoculture, apoptosis induction was accompanied by downregulation of the NF-κB target protein TRAF1 (untreated vs. treated: expression reduced by 34 %, p=0,0044), upregulation of the proapoptotic protein BAX (expression increased by 3175 %, p=0,0268), and increased PARP cleavage (100% vs. 8393% expression, p=0,0078). Conversely, in co-culture, downregulation of TRAF1 by 52 % (p=0,0054) was observed without concomitant BAX upregulation or PARP cleavage matching the non-appearance of apoptosis induction in those cells. While co-culturing untreated CLL cells on BMSC led to tendentially increased expression levels of the non-canonical NF-κB subunits NF-κB2 (untreated monocultured cells vs. untreated co-cultured cells: 100% vs. 142%, p=0,8438) and RELB (100% vs. 128%, p=0,7422), NF-κB DNA-binding activities of all NF-κB subunits were equally suppressed by DHMEQ treatment in mono- and co-cultured cells (e.g. treated monocultured vs. treated co-cultured cells: 1,6% vs. 4,9%, p<0,9999 for NF-κB1). Gene knockdown of the NF-κB subunit RELA by siRNA transfection solely induced apoptosis in monocultured CLL cells as well. Adding soluble BAFF to monocultured treated CLL cells attenuated DHMEQ efficiency (viability 1,3% vs. 16%, p=0,0258, n=9), while adding APRIL, CD40 ligand and SDF-1a had little influence on the response to treatment. Finally, the combined use of DHMEQ with fludarabine in co-cultured CLL cells led to a higher rate of apoptosis than DHMEQ (viability 57% vs. 37%, p=0.0202) or fludarabine alone (viability 50% vs. 37%, p=0.1828). Conclusion: NF-κB inhibition in primary CLL cells shows great discrepancy between in vitro and in vivo scenarios. While DHMEQ treatment leads to apoptosis in mono-cultured cells by BAX upregulation and increased PARP cleavage, CLL cell viability is not affected in the presence of microenvironment, suggesting that the NF-κB pathway can be bypassed in vivo. Soluble ligands, especially BAFF, appear to be involved in mediating this protective effect. However, the combination of NF-κB inhibition with standard chemotherapy might represent a promising approach and warrants further clinical assessment. Disclosures No relevant conflicts of interest to declare.

scholarly journals Retinoid-induced apoptosis and Sp1 cleavage occur independently of transcription and require caspase activation.

1997 ◽  
Vol 17 (11) ◽  
pp. 6348-6358 ◽  
Author(s):  
F J Piedrafita ◽  
M Pfahl
Keyword(s):  
T Cells ◽  
Dna Binding ◽  
Cancer Cell ◽  
De Novo ◽  
Binding Activity ◽  
Dependent Manner ◽  
Intact Cells ◽  
Dna Binding Activity ◽  
Induced Apoptosis

Vitamin A and its derivatives, the retinoids, are essential regulators of many important biological functions, including cell growth and differentiation, development, homeostasis, and carcinogenesis. Natural retinoids such as all-trans retinoic acid can induce cell differentiation and inhibit growth of certain cancer cells. We recently identified a novel class of synthetic retinoids with strong anti-cancer cell activities in vitro and in vivo which can induce apoptosis in several cancer cell lines. Using an electrophoretic mobility shift assay, we analyzed the DNA binding activity of several transcription factors in T cells treated with apoptotic retinoids. We found that the DNA binding activity of the general transcription factor Sp1 is lost in retinoid-treated T cells undergoing apoptosis. A truncated Sp1 protein is detected by immunoblot analysis, and cytosolic protein extracts prepared from apoptotic cells contain a protease activity which specifically cleaves purified Sp1 in vitro. This proteolysis of Sp1 can be inhibited by N-ethylmaleimide and iodoacetamide, indicating that a cysteine protease mediates cleavage of Sp1. Furthermore, inhibition of Sp1 cleavage by ZVAD-fmk and ZDEVD-fmk suggests that caspases are directly involved in this event. In fact, caspases 2 and 3 are activated in T cells after treatment with apoptotic retinoids. The peptide inhibitors also blocked retinoid-induced apoptosis, as well as processing of caspases and proteolysis of Sp1 and poly(ADP-ribose) polymerase in intact cells. Degradation of Sp1 occurs early during apoptosis and is therefore likely to have profound effects on the basal transcription status of the cell. Interestingly, retinoid-induced apoptosis does not require de novo mRNA and protein synthesis, suggesting that a novel mechanism of retinoid signaling is involved, triggering cell death in a transcriptional activation-independent, caspase-dependent manner.

Degradation of constitutive transcription factors during apoptosis in rat thymocytes

1994 ◽  
Vol 72 (11-12) ◽  
pp. 639-648 ◽  
Author(s):  
Ian de Belle ◽  
Lucia Testolin ◽  
Siyaram Pandey ◽  
Christine Carson ◽  
P. Roy Walker ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcription Factors ◽  
Dna Binding ◽  
Binding Proteins ◽  
Dna Binding Proteins ◽  
Binding Activity ◽  
Significant Loss ◽  
Dna Binding Activity ◽  
The Stability ◽  
Induced Apoptosis

Dexamethasone (Dex) accelerates the rate of apoptosis in thymocytes by a process thought to require gene expression. Among the genes implicated in the regulation of this phenomenon are the immediate early genes such as c-fos and c-jun, whose expression is modulated by a complement of preexisting transcription factors. We have analyzed the DNA-binding activity of these constitutive transcription factors during Dex-induced apoptosis in thymocytes to assess their functionality. We observed a progressive loss of the DNA-binding proteins in parallel with the appearance of the characteristic morphological and biochemical features of apoptosis. At the same time we have found a general increase in the nuclear proteolytic activity concomitant with a significant loss of the nuclear nonhistone chromosomal proteins. Indeed, cotreatment of thymocytes with the nonspecific serine protease inhibitor phenyl-methylsulphonyl fluoride was able to partially protect the stability of the DNA-binding proteins and alter the expression of the c-fos and c-jun genes but did not inhibit apoptosis. Our results suggest that the action of a protease(s) is responsible for the degradation of constitutive transcription factors during Dex-induced apoptosis, rendering the death pathway irreversible.Key words: apoptosis, thymocytes, proteolysis, transcription factors, gene expression.

Gold Compound Auranofin (RIDAURA®) Induces Apoptosis of Human Myeloma Cells by Targeting STAT3 and NF-κB Pathways, with Clinical Potential.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3837-3837 ◽  
Author(s):  
Morihiko Sagawa ◽  
Aya Nakaya ◽  
Tatsuki Tomikawa ◽  
Yuichiro Haba ◽  
Tomoe Nemoto ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
Multiple Myeloma ◽  
Drug Resistance ◽  
Dna Binding ◽  
Clinical Outcome ◽  
Binding Activity ◽  
Gold Compound ◽  
Constitutive Activation ◽  
Dna Binding Activity ◽  
Induced Apoptosis

Abstract Abstract 3837 Poster Board III-773 Multiple myeloma (MM) is a plasma cell malignancy that remains incurable despite the use of conventional chemotherapy. Recent advances in the field of cell biology could lead to new therapeutic agents based upon molecular targeting, such as thalidomide and proteasome inhibitor bortezomib. These agents have remarkable activity against both newly diagnosed and refractory patients with MM, but prolonged exposure to them may result in the development of de novo drug resistance. Thus, it is necessary to identify and validate novel agents with less toxicity to overcome drug resistance and to improve the clinical outcome of MM. Progression and chemoresistance are thought to involve IL-6, whose expression is induced by NF-κB, through its regulation of the growth and survival of MM cells. IL-6 leads to constitutive activation of STAT3, which in turn results in the expression of high levels of Mcl-1. Thus, the constitutive activation of both NF-κB and STAT3 plays an important role in MM cell proliferation, and both molecules are important targets for the treatment of MM. Auranofin (AF: RIDAURA®; GSK) is a coordinated gold compound that has been widely used for the treatment of rheumatoid arthritis based on its anti-inflammatory properties through the inhibition of NF-κB activation. Therefore, assuming that AF has the potency to induce apoptosis in MM cells by interfering with NF-κB and STAT pathways, it may become a candidate for a novel therapeutic agent. To address our hypothesis, the effects of AF on inducing apoptosis of various MM cells were examined. Further, the molecular mechanism of AF-induced apoptosis in MM cells was investigated. AF inhibited the growth of U266 cells in a time- and dose-dependent manner with IC50 of 50 nM at 24 h. AF significantly induced cell cycle arrest at the G1 phase and subsequent apoptosis of U266 cells. AF-induced apoptosis in various human MM cell lines and CD138-positive plasma cells from patients with MM involved the activation of caspases-3, -8, and -9. Treatment with AF inhibited the constitutive and IL-6-induced activation of STAT3, and then downregulated the expression of Mcl-1 but not that of Bcl-2 or Bcl-xL proteins. To clarify the biological significance of Mcl-1 in AF-induced apoptosis of MM cells, Mcl-1 expression vector (pEGFP-hmcl-1) and control vector were introduced into U266 cells (designated as U266/mcl and U266/neo cells). Induction of apoptosis by AF was abrogated in U266/mcl, but not in U266/neo cells. We next examined the effect of AF on the DNA binding activity of STAT3. Electrophoretic mobility gel shift assay (EMSA) using U266 nuclear extracts demonstrated that IL-6-induced STAT3 binding activity was inhibited by the presence of AF. These results suggest that AF inhibits the IL-6-induced JAK/STAT pathway selectively and induces apoptosis in MM cells via the downregulation of Mcl-1. In addition, AF downregulated the activation of NF-κB in an IκB-independent manner, and also inhibited DNA binding activity in U266 cells. Although NF-κB inhibitory peptide (SN-50) did not directly induce the expression of Mcl-1, the combination of SN-50 with AF reduced the levels of Mcl-1, suggesting that inhibition of NF-κB potentiates the apoptotic effect of AF. Finally, our in vitro data prompted us to examine whether or not the effects of AF are equally valid in a clinical settings. The protocol for assessing the clinical benefit of oral administration of AF (RIDAURA®) in patients with relapsed or refractory MM was approved by the Ethics Committee of Saitama Medical University. Since 2008, 7 patients with refractory MM were treated with oral RIDAURA® 6 mg daily. The median follow-up for patients was 7.5 months. All 7 patients responded to AF, including one PR (partial response) and 6 SD (stable disease) with no progression. No adverse events have been observed to date. Details of the clinical outcome will be presented. In conclusion, gold compound AF inhibited constitutive activation of both STAT3 and NF-κB, resulting in the downregulation of anti-apoptotic Mcl-1 protein in MM cells with clinical relevance. A low pharmacological concentration (50 nM) of AF is widely employed for the treatment of rheumatoid arthritis without any side effects; therefore, it may be used to treat MM without the risk of severe toxicity. We propose that AF (RIDAURA®) may have potential use as a new molecular-targeted agent for the treatment of MM. Disclosures: Off Label Use: Auranofin in the treatment of multiple myeloma.

scholarly journals Disruption of Microtubules Sensitizes the DNA Damage-induced Apoptosis Through Inhibiting Nuclear Factor κB (NF-κB) DNA-binding Activity

2010 ◽  
Vol 25 (11) ◽  
pp. 1574 ◽  
Author(s):  
Hyunji Lee ◽  
Juhee Jeon ◽  
Young Sue Ryu ◽  
Jae Eun Jeong ◽  
Sanghee Shin ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Binding ◽  
Nuclear Factor Κb ◽  
Binding Activity ◽  
Nuclear Factor ◽  
Dna Binding Activity ◽  
Induced Apoptosis

Dynamic Changes in the Composition of the AP-1 Transcription Factor DNA-binding Activity in Rat Brain Following Kainate-induced Seizures and Cell Death

1994 ◽  
Vol 6 (10) ◽  
pp. 1558-1566 ◽  
Author(s):  
Bozena Kaminska ◽  
Robert K. Filipkowski ◽  
Grazyna Zurkowska ◽  
Wladyslaw Lason ◽  
Ryszard Przewlocki ◽  
...  
Keyword(s):  
Cell Death ◽  
Transcription Factor ◽  
Dna Binding ◽  
Rat Brain ◽  
Binding Activity ◽  
Dynamic Changes ◽  
Dna Binding Activity
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