benzyloxycarbonylvalyl-alanyl-aspartyl-fluoromethyl-ketone and Hematologic-Neoplasms

We have previously reported that an anti-human transferrin receptor IgG3-avidin fusion protein (anti-hTfR IgG3-Av) inhibits the proliferation of an erythroleukemia-cell line. We have now found that anti-hTfR IgG3-Av also inhibits the proliferation of additional human malignant B and plasma cells. Anti-hTfR IgG3-Av induces internalization and rapid degradation of the TfR. These events can be reproduced in cells treated with anti-hTfR IgG3 cross-linked with a secondary Ab, suggesting that they result from increased TfR cross-linking. Confocal microscopy of cells treated with anti-hTfR IgG3-Av shows that the TfR is directed to an intracellular compartment expressing the lysosomal marker LAMP-1. The degradation of TfR is partially blocked by cysteine protease inhibitors. Furthermore, cells treated with anti-hTfR IgG3-Av exhibit mitochondrial depolarization and activation of caspases 9, 8, and 3. The mitochondrial damage and cell death can be prevented by iron supplementation, but cannot be fully blocked by a pan-caspase inhibitor. These results suggest that anti-hTfR IgG3-Av induces lethal iron deprivation, but the resulting cell death does not solely depend on caspase activation. This report provides insights into the mechanism of cell death induced by anti-TfR Abs such as anti-hTfR IgG3-Av, a molecule that may be useful in the treatment of B-cell malignancies such as multiple myeloma.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Avidin; Caspase Inhibitors; Cell Line, Tumor; Cell Proliferation; Cross-Linking Reagents; Deferoxamine; Hematologic Neoplasms; Humans; Immunoglobulin G; Iron; Leukemia, Plasma Cell; Multiple Myeloma; Receptors, Transferrin; Recombinant Fusion Proteins; Siderophores

Diverse death stimuli including anticancer drugs trigger apoptosis by inducing the translocation of cytochrome c from the outer mitochondrial compartment into the cytosol. Once released, cytochrome c cooperates with apoptotic protease-activating factor-1 and deoxyadenosine triphosphate in caspase-9 activation and initiation of the apoptotic protease cascade. The results of this study show that on death induction by chemotherapeutic drugs, staurosporine and triggering of the death receptor CD95, cytochrome c not only translocates into the cytosol, but furthermore can be abundantly detected in the extracellular medium. The cytochrome c release from the cell is a rapid and apoptosis-specific process that occurred within 1 hour after induction of apoptosis, but not during necrosis. Interestingly, elevated cytochrome c levels were observed in sera from patients with hematologic malignancies. In the course of cancer chemotherapy, the serum levels of cytochrome c in the majority of the patients grew rapidly as a result of increased cell death. These data suggest that monitoring of cytochrome c in the serum of patients with tumors might serve as a useful clinical marker for the detection of the onset of apoptosis and cell turnover in vivo.

Topics: Adult; Aged; Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Biomarkers; Biomarkers, Tumor; Chick Embryo; Cysteine Proteinase Inhibitors; Cytochrome c Group; Cytosol; Dactinomycin; Enzyme Inhibitors; Extracellular Space; Female; Hematologic Neoplasms; Humans; Hydrogen Peroxide; Jurkat Cells; L Cells; L-Lactate Dehydrogenase; Male; Mice; Middle Aged; Mitochondria; Necrosis; Neoplasm Proteins; Nucleic Acid Synthesis Inhibitors; Protein Transport; Staurosporine; Tumor Necrosis Factor-alpha

The recombinant immunotoxins anti-Tac(Fv)-PE38 (LMB-2), targeting the interleukin-2 receptor alpha subunit (IL-2Ralpha, Tac or CD25), and RFB4(dsFv)-PE38 (BL22), targeting CD22, are being evaluated in clinical trials as treatment for hematologic malignancies. The toxin moiety Pseudomonas exotoxin A (PE) of these recombinant molecules leads to the arrest of protein synthesis due to inactivation of elongation factor 2. Here, we provide evidence that cell lines derived from patients with hematologic malignancies react to immunotoxins not only with inhibition of protein synthesis but also with characteristic hallmarks of apoptosis such as caspase activation, cleavage of the "death substrate poly(ADP)-ribose polymerase and DNA laddering. Anti-Tac(Fv)-PE38 leads to a 10-fold increase in the cleavage of the fluorescent substrate DEVD-AFC, suggesting that a caspase-3-like enzyme is involved. This was verified by cleavage of caspase-3 (CPP32). MT1 cells exhibited DNA laddering after treatment with immunotoxin, which was reversed by pre-treatment with the protease inhibitor zVAD-fmk. This caspase inhibitor led to an at least 5-fold improvement in cell viability without altering inhibition of protein synthesis. Interestingly, HUT-102 cells did not undergo programmed cell death after exposure to immunotoxins that kill these cells. We conclude that immunotoxins may be valuable in the treatment of cancers that are resistant toward apoptosis because their targeted killing is often facilitated by, but not completely dependent on, programmed cell death. Int. J. Cancer 87:86-94, 2000. Published 2000 Wiley-Liss, Inc.

Topics: Amino Acid Chloromethyl Ketones; Antibodies; Antigens, CD; Antigens, Differentiation, B-Lymphocyte; Apoptosis; Burkitt Lymphoma; Caspase 3; Caspases; Cell Adhesion Molecules; Cell Survival; Coumarins; DNA Fragmentation; Dose-Response Relationship, Drug; Hematologic Neoplasms; Immunoblotting; Immunotoxins; Lectins; Leucine; Leukemia; Lymphoma, T-Cell; Oligopeptides; Protease Inhibitors; Recombinant Proteins; Sialic Acid Binding Ig-like Lectin 2; Tetrazolium Salts; Tumor Cells, Cultured