Apoptosis of haematopoietic cells upon thymidylate synthase inhibition is independent of p53 accumulation and CD95–CD95 ligand interaction

2000 ◽  
Vol 353 (1) ◽  
pp. 101-108 ◽  
Author(s):  
Cristina MUÑOZ-PINEDO ◽  
F. Javier OLIVER ◽  
Abelardo LÓPEZ-RIVAS
Keyword(s):  
Thymidylate Synthase ◽  
Virus Protein ◽  
Protein Accumulation ◽  
Drug Induced ◽  
Ligand Interaction ◽  
Cd95 Ligand ◽  
Marked Accumulation ◽  
Induced Apoptosis ◽  
Synthase Inhibitor ◽  
In Cells

Treatment of haematopoietic BA/F3 cells with the thymidylate synthase inhibitor 5-fluoro-2′-deoxyuridine (FUdR) activated apoptosis through a mechanism that required continuous protein synthesis and was inhibited by Bcl-2 over-expression. Analysis of p53 levels in cells treated with FUdR indicated a marked accumulation of this protein. Accumulation of p53 was also observed in cells over-expressing Bcl-2. In BA/F3 cells transfected with a cDNA coding for the human papilloma virus protein E6, p53 accumulation after FUdR treatment was inhibited markedly. However, apoptosis was induced in both control and E6 cells to a similar extent. The role of the CD95/CD95 ligand (CD95L) system in FUdR-induced apoptosis was also assessed. As determined by reverse transcriptase PCR, BA/F3 expressed a low constitutive level of CD95L mRNA, which decreased following FUdR treatment. Moreover, blocking CD95–CD95L interactions with antagonistic CD95 monoclonal antibody did not prevent drug-induced apoptosis. Furthermore, analysis of caspase involvement showed important differences in apoptosis induced by CD95-triggering or FUdR treatment. In summary, these results suggest that apoptosis induced by thymineless stress in haematopoietic BA/F3 cells occurs by a mechanism that does not require accumulation of p53 and which is independent of CD95–CD95L interactions.

Functional CD95 ligand and CD95 death-inducing signaling complex in activation-induced cell death and doxorubicin-induced apoptosis in leukemic T cells

Blood ◽  
2000 ◽  
Vol 95 (1) ◽  
pp. 301-308 ◽  
Author(s):  
Simone Fulda ◽  
Gudrun Strauss ◽  
Eric Meyer ◽  
Klaus-Michael Debatin
Keyword(s):  
T Cells ◽  
Cell Death ◽  
Leukemia Cell Line ◽  
Receptor Interaction ◽  
Caspase 8 ◽  
Drug Induced ◽  
Signaling Complex ◽  
Cd95 Ligand ◽  
Activation Induced Cell Death ◽  
Induced Apoptosis

Abstract Activation-induced cell death (AICD) in T cells is mediated by CD95 ligand (CD95L)/receptor interaction, which has also been implicated in apoptosis induction by some anticancer agents. In this article we show that both anti-CD3-triggering (AICD) and doxorubicin treatment led to the production of a functionally active CD95L in the CD3+/T-cell receptor-positive (TCR+) T leukemia cell line H9. CD95L-expressing H9 cells killed CD95-sensitive J16 or CEM target cells, but not CD95-resistant CEM or J16 cells overexpressing dominant negative FADD (J16/FADD-DN). By immunoprecipitation, CD95L was physically bound to CD95, suggesting that AICD and doxorubicin-induced apoptosis involve CD95L-mediated CD95 aggregation, thereby triggering the CD95 death pathway. CD95 aggregation was associated with the recruitment of FADD and caspase-8 to the CD95 receptor to form the CD95 death-inducing signaling complex (DISC), resulting in caspase-8 activation and cleavage of the effector caspase-3 and PARP. Blocking of the CD95L/receptor interaction by antagonistic antibodies to CD95 or to CD95L also blocked AICD and inhibited the early phase of doxorubicin-induced apoptosis, though cell death induced by doxorubicin eventually proceeded in a CD95-independent manner. These findings may explain some conflicting data on the role of death receptor systems in drug-induced apoptosis. Thus, in cells with an inducible CD95 receptor/ligand system, drug-induced apoptosis may be mediated by CD95L-initiated DISC formation and activation of downstream effector programs similar to AICD in T cells. (Blood. 2000;95:301-308)

Functional CD95 ligand and CD95 death-inducing signaling complex in activation-induced cell death and doxorubicin-induced apoptosis in leukemic T cells

Blood ◽  
2000 ◽  
Vol 95 (1) ◽  
pp. 301-308 ◽  
Author(s):  
Simone Fulda ◽  
Gudrun Strauss ◽  
Eric Meyer ◽  
Klaus-Michael Debatin
Keyword(s):  
T Cells ◽  
Cell Death ◽  
Leukemia Cell Line ◽  
Receptor Interaction ◽  
Caspase 8 ◽  
Drug Induced ◽  
Signaling Complex ◽  
Cd95 Ligand ◽  
Activation Induced Cell Death ◽  
Induced Apoptosis

Activation-induced cell death (AICD) in T cells is mediated by CD95 ligand (CD95L)/receptor interaction, which has also been implicated in apoptosis induction by some anticancer agents. In this article we show that both anti-CD3-triggering (AICD) and doxorubicin treatment led to the production of a functionally active CD95L in the CD3+/T-cell receptor-positive (TCR+) T leukemia cell line H9. CD95L-expressing H9 cells killed CD95-sensitive J16 or CEM target cells, but not CD95-resistant CEM or J16 cells overexpressing dominant negative FADD (J16/FADD-DN). By immunoprecipitation, CD95L was physically bound to CD95, suggesting that AICD and doxorubicin-induced apoptosis involve CD95L-mediated CD95 aggregation, thereby triggering the CD95 death pathway. CD95 aggregation was associated with the recruitment of FADD and caspase-8 to the CD95 receptor to form the CD95 death-inducing signaling complex (DISC), resulting in caspase-8 activation and cleavage of the effector caspase-3 and PARP. Blocking of the CD95L/receptor interaction by antagonistic antibodies to CD95 or to CD95L also blocked AICD and inhibited the early phase of doxorubicin-induced apoptosis, though cell death induced by doxorubicin eventually proceeded in a CD95-independent manner. These findings may explain some conflicting data on the role of death receptor systems in drug-induced apoptosis. Thus, in cells with an inducible CD95 receptor/ligand system, drug-induced apoptosis may be mediated by CD95L-initiated DISC formation and activation of downstream effector programs similar to AICD in T cells. (Blood. 2000;95:301-308)

scholarly journals Anticancer Drugs Induce Caspase-8/FLICE Activation and Apoptosis in the Absence of CD95 Receptor/Ligand Interaction

Blood ◽  
1999 ◽  
Vol 93 (9) ◽  
pp. 3053-3063 ◽  
Author(s):  
Sebastian Wesselborg ◽  
Ingo H. Engels ◽  
Evi Rossmann ◽  
Marek Los ◽  
Klaus Schulze-Osthoff
Keyword(s):  
Anticancer Drugs ◽  
De Novo ◽  
Death Receptor ◽  
Dominant Negative ◽  
Caspase 8 ◽  
Drug Induced ◽  
Ligand Interaction ◽  
Caspase Family ◽  
Death Receptor Signaling ◽  
Induced Apoptosis

Abstract Proteases of the caspase family are the critical executioners of apoptosis. Their activation has been mainly studied upon triggering of death receptors, such as CD95 (Fas/APO-1) and tumor necrosis factor-R1, which recruit caspase-8/FLICE as the most proximal effector to the receptor complex. Because apoptosis induced by anticancer drugs has been proposed to involve CD95/CD95 ligand interaction, we investigated the mechanism of caspase activation by daunorubicin, doxorubicin, etoposide, and mitomycin C. In Jurkat leukemic T cells, all drugs induced apoptosis and the cleavage of procaspase-8 to its active p18 subunit. However, cells resistant to CD95 were equally susceptible to anticancer drugs and activated caspase-8 with a similar kinetic and dose response as CD95-sensitive cells. The broad caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone prevented apoptosis and caspase-8 activation in response to CD95 and drug treatment, whereas a neutralizing CD95 decoy as well as a dominant-negative FADD construct selectively abrogated CD95, but not drug-induced effects. A potent activation of caspase-8 was also induced by cycloheximide, indicating that it was independent of protein synthesis. Our data, therefore, show that (1) anticancer drug-induced apoptosis does not require de novo synthesis of death ligands or CD95 interaction, and (2) that caspase-8 can be activated in the absence of a death receptor signaling.

scholarly journals A Novel AP-1 Element in the CD95 Ligand Promoter Is Required for Induction of Apoptosis in Hepatocellular Carcinoma Cells upon Treatment with Anticancer Drugs

2000 ◽  
Vol 20 (20) ◽  
pp. 7826-7837 ◽  
Author(s):  
Sören T. Eichhorst ◽  
Martina Müller ◽  
Min Li-Weber ◽  
Henning Schulze-Bergkamen ◽  
Peter Angel ◽  
...  
Keyword(s):  
Carcinoma Cells ◽  
Tata Box ◽  
Chemotherapeutic Drugs ◽  
Drug Induced ◽  
Anticancer Chemotherapy ◽  
Extracellular Signals ◽  
Cd95 Ligand ◽  
Hepatocellular Carcinomas ◽  
Induction Of Apoptosis ◽  
Induced Apoptosis

ABSTRACT The CD95 (also called APO-1 or Fas) system plays a major role in the induction of apoptosis in lymphoid and nonlymphoid tissues in response to a variety of extracellular signals, including chemotherapeutic drugs. Here we report that the CD95 ligand (CD95L) is upregulated in hepatoma cells upon treatment with antineoplastic drugs. Upregulation by different chemotherapeutic drugs is functionally relevant for drug-induced apoptosis and is mediated by transcriptional mechanisms. The MEKK1/JNKK pathway and a novel AP-1 element in the CD95L promoter downstream of the TATA box are required for CD95L upregulation. Thus, understanding the mechanisms of CD95-mediated apoptosis through CD95L upregulation upon treatment of hepatocellular carcinomas with chemotherapeutic drugs may contribute to the improvement of anticancer chemotherapy.

scholarly journals Anticancer Drugs Induce Caspase-8/FLICE Activation and Apoptosis in the Absence of CD95 Receptor/Ligand Interaction

Blood ◽  
1999 ◽  
Vol 93 (9) ◽  
pp. 3053-3063 ◽  
Author(s):  
Sebastian Wesselborg ◽  
Ingo H. Engels ◽  
Evi Rossmann ◽  
Marek Los ◽  
Klaus Schulze-Osthoff
Keyword(s):  
Anticancer Drugs ◽  
De Novo ◽  
Death Receptor ◽  
Dominant Negative ◽  
Caspase 8 ◽  
Drug Induced ◽  
Ligand Interaction ◽  
Caspase Family ◽  
Death Receptor Signaling ◽  
Induced Apoptosis

Proteases of the caspase family are the critical executioners of apoptosis. Their activation has been mainly studied upon triggering of death receptors, such as CD95 (Fas/APO-1) and tumor necrosis factor-R1, which recruit caspase-8/FLICE as the most proximal effector to the receptor complex. Because apoptosis induced by anticancer drugs has been proposed to involve CD95/CD95 ligand interaction, we investigated the mechanism of caspase activation by daunorubicin, doxorubicin, etoposide, and mitomycin C. In Jurkat leukemic T cells, all drugs induced apoptosis and the cleavage of procaspase-8 to its active p18 subunit. However, cells resistant to CD95 were equally susceptible to anticancer drugs and activated caspase-8 with a similar kinetic and dose response as CD95-sensitive cells. The broad caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone prevented apoptosis and caspase-8 activation in response to CD95 and drug treatment, whereas a neutralizing CD95 decoy as well as a dominant-negative FADD construct selectively abrogated CD95, but not drug-induced effects. A potent activation of caspase-8 was also induced by cycloheximide, indicating that it was independent of protein synthesis. Our data, therefore, show that (1) anticancer drug-induced apoptosis does not require de novo synthesis of death ligands or CD95 interaction, and (2) that caspase-8 can be activated in the absence of a death receptor signaling.

Activation of caspase-8 in drug-induced apoptosis of B-lymphoid cells is independent of CD95/Fas receptor-ligand interaction and occurs downstream of caspase-3

Blood ◽  
2001 ◽  
Vol 97 (5) ◽  
pp. 1378-1387 ◽  
Author(s):  
Thomas Wieder ◽  
Frank Essmann ◽  
Aram Prokop ◽  
Karin Schmelz ◽  
Klaus Schulze-Osthoff ◽  
...  
Keyword(s):  
Caspase 3 ◽  
Death Receptor ◽  
Lymphoid Cells ◽  
Caspase 8 ◽  
Lymphoma Cells ◽  
Drug Induced ◽  
Ligand Interaction ◽  
B Lymphoma ◽  
B Lymphoma Cells ◽  
Induced Apoptosis

The activation of caspase-8, a crucial upstream mediator of death receptor signaling, was investigated in epirubicin- and Taxol-induced apoptosis of B-lymphoma cells. This study was performed because the CD95/Fas receptor-ligand interaction, recruitment of the Fas-associated death domain (FADD) adaptor protein, and subsequent activation of procaspase-8 have been implicated in the execution of drug-induced apoptosis in other cell types. Indeed, active caspase-8 was readily detected after treatment of mature and immature B-lymphoid cells with epirubicin or Taxol. However, neither constitutive nor drug-induced expression of the CD95/Fas ligand was detectable in B-lymphoma cells. Furthermore, overexpression of a dominant-negative FADD mutant (FADDdn) did not block caspase-8 processing and subsequent DNA fragmentation, indicating that drug-induced caspase-8 activation was mediated by a CD95/Fas-independent mechanism. Instead, caspase-8 cleavage was slightly preceded by activation of caspase-3, suggesting that drug-induced caspase-8 activation in B-lymphoma cells is a downstream event mediated by other caspases. This assumption was confirmed in 2 experimental systems—zDEVD-fmk, a cell-permeable inhibitor of caspase-3–like activity, blocked drug-induced caspase-8 cleavage, and depletion of caspase-3 from cell extracts impaired caspase-8 cleavage after in vitro activation with dATP and cytochrome c. Thus, these data indicate that drug-induced caspase-8 activation in B-lymphoma cells is independent of death receptor signaling and is mediated by postmitochondrial caspase-3 activation.

Pomolic Acid-Induced Apoptosis in Cells from Chronic Myeloid Leukemic Patients Is Not Affected by the MDR Phenotype and Status of Disease.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4889-4889
Author(s):  
Flavia C. Vasconcelos ◽  
Cerli R. Gattas ◽  
Janaina Fernandes ◽  
Vivian M.D. Rumjanek ◽  
Raquel C. Maia
Keyword(s):  
Chronic Phase ◽  
Annexin V ◽  
Leukemic Cells ◽  
Drug Induced ◽  
Imatinib Resistance ◽  
Blastic Phase ◽  
Pomolic Acid ◽  
Apoptotic Effect ◽  
Induced Apoptosis ◽  
In Cells

Abstract Pomolic acid (PA), isolated from Chrysobalanus icaco, is a pentaciclic triterpene highly effective in inhibiting the growth and promotes cell death in the human chronic myeloid leukemia (CML) K562 cell line. In addition, PA is also very effective in blocking the proliferation of K562-Lucena-1, a vincristine-resistant derivative of K562 that displays multidrug resistance (MDR) phenotype. Due to antiproliferative and antiapoptotic effect of PA on positive and negative MDR leukemic cells, and considering that some patients with CML exhibit resistance to imatinib, the aim of this work was to investigate if PA could have an effective role in induce apoptosis in cells from CML patients. Fourteen peripheral blood samples from CML patients in various stages (three in blastic phase and eleven in chronic phase being one juvenile patient) including previously clinically imatinib resistant patients were studied. Informed consent was obtained according to institutional guidelines. Cells from patients were plated in 96 well tissue culture plates for 24h and then treated with two different concentrations of PA: 12.5 μg/ml and 25 μg/ml. Drug-induced apoptosis was evaluated by annexin V/propidium iodide assessed by fluorescence-activated cell-sorting (FACS). Functional MDR phenotype was performed by measuring the fluorescent dye rhodamine-123 efflux in the presence or absence of the MDR blocker cyclosporin A. These experiments were analysed by FACS. Functional activity of MDR was observed in 9 out of 14 samples and it was independent of CML stage. Using 12.5 μg/ml and 25μg/ml concentrations, the apoptotic effect of PA ranged from 10.5 to 53.1 (median: 31.5) and 14.7 to 65.9 (median: 42.2), respectively. These results were also independent of CML stage being even effective in blastic phase and in juvenile CML, which have a clinically aggressive course. Our data demonstrate that PA is a potent anti-leukemic agent not only for MDR positive CML patients but also especially for the blastic phase in the setting of imatinib resistance.

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