Involvement of p38 Activation and Mitochondria in Death of Human Leukemia Cells Induced by an Agonistic Human Monoclonal Antibody Fab Specific to TRAIL Receptor 1
Abstract
:1. Introduction
2. Results
2.1. Characterization of a Human mAb Fab against DR4
2.2. DR4-4 Induces Cell Death in Various Cancer Cells
2.3. DR4-4 Fab Induces Caspase-Independent Cell Death
2.4. Mitochondria Are Involved in DR4-4 Fab–Induced Death
2.5. DR4-4 Fab Activates p38 MAPK, Inhibits c-FLIP, and Increases Cell Death Rates in Combination with TRAIL or γ-Irradiation
3. Discussion
4. Materials and Methods
4.1. Reagents
4.2. Phage Display and Purification of Soluble Fab
4.3. Cell Culture
4.4. Immunoblotting
4.5. Direct Binding and Competitive ELISA
4.6. Affinity Analysis by SPR
4.7. FACS Analysis for Cellular Binding of DR4-4 Fab
4.8. Cytotoxicity Test
4.9. Mitochondrial ROS and MMP Measurement
4.10. Confocal Microscopy for Mitochondrial Morphology
4.11. Electron Microscopy for Cellular Image Analysis
4.12. γ-Irradiation
4.13. Statistical Analysis
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
Abbreviations
Ab | antibody |
BAPTA | 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid |
Bcl-2 | B-cell lymphoma 2 |
Bcl-xL | B-cell lymphoma extra-large |
Bid | BH3 interacting-domain death agonist |
c-FLIP | cellular FADD-like IL-1β-converting enzyme–inhibitory protein |
DcR | decoy receptor |
DISC | death-inducing signaling complex |
DR | death receptor |
ELISA | enzyme-linked immunosorbent assay |
ERK | extracellular signal-regulated kinase |
Fab | antigen binding fragment |
FACS | fluorescence-activated cell sorting |
FITC | fluorescein isothiocyanate |
IκBα | nuclear factor κB inhibitor α |
JNK | c-Jun N-terminal kinase |
mAb | monoclonal antibody |
Mcl-1 | myeloid cell leukemia 1 |
MMP | mitochondrial membrane potential |
MTT | 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide |
NAC | N-acetyl-l-cysteine |
NF-κB | nuclear factor κB |
p38 MAPK | p38 mitogen-activated protein kinase |
PBS | phosphate-buffered saline |
ROS | reactive oxygen species |
SPR | surface plasmon resonance |
sTRAIL | soluble TRAIL |
tBid | truncated Bid |
TNF | tumor necrosis factor |
TRAIL | tumor necrosis factor–related apoptosis-inducing ligand |
VH | heavy chain |
VL | light chain |
WM | wortmannin |
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Lee, Y.-R.; Hwang, E.; Jang, Y.-J. Involvement of p38 Activation and Mitochondria in Death of Human Leukemia Cells Induced by an Agonistic Human Monoclonal Antibody Fab Specific to TRAIL Receptor 1. Int. J. Mol. Sci. 2019, 20, 1967. https://doi.org/10.3390/ijms20081967
Lee Y-R, Hwang E, Jang Y-J. Involvement of p38 Activation and Mitochondria in Death of Human Leukemia Cells Induced by an Agonistic Human Monoclonal Antibody Fab Specific to TRAIL Receptor 1. International Journal of Molecular Sciences. 2019; 20(8):1967. https://doi.org/10.3390/ijms20081967
Chicago/Turabian StyleLee, You-Ri, Eunjoo Hwang, and Young-Ju Jang. 2019. "Involvement of p38 Activation and Mitochondria in Death of Human Leukemia Cells Induced by an Agonistic Human Monoclonal Antibody Fab Specific to TRAIL Receptor 1" International Journal of Molecular Sciences 20, no. 8: 1967. https://doi.org/10.3390/ijms20081967