ABSTRACT
We studied the actions of 2-phenylacetylenesulfonamide (PAS) on B-chronic lymphocytic leukemia (CLL) cells. PAS (5-20 microM) initiated apoptosis within 24 hours, with maximal death at 48 hours asassessed by morphology, cleavage of poly(ADP-ribose) polymerase (PARP), caspase 3 activation, and annexin V staining. PAS treatment induced Bax proapoptotic conformational change, Bax movement from the cytosol to the mitochondria, and cytochrome c release, indicating that PAS induced apoptosis via the mitochondrial pathway. PAS induced approximately 3-fold up-regulation of proapoptotic Noxa protein and mRNA levels. In addition, Noxa was found unexpectedly to be bound to Bcl-2 in PAS-treated cells. PAS treatment of CLL cells failed to up-regulate p53, suggesting that PAS induced apoptosis independently of p53. Furthermore, PAS induced apoptosis in CLL isolates with p53 gene deletion in more than 97% of cells. Normal B lymphocytes were as sensitive to PAS-induced Noxa up-regulation and apoptosis as were CLL cells. However, both T lymphocytes and bone marrow hematopoietic progenitor cells were relatively resistant to PAS. Our data suggest that PAS may represent a novel class of drug that induces apoptosis in CLL cells independently of p53 status by a mechanism involving Noxa up-regulation.
Subject(s)
Antineoplastic Agents/pharmacokinetics , Apoptosis/drug effects , Gene Expression Regulation, Leukemic/drug effects , Leukemia, Lymphocytic, Chronic, B-Cell/drug therapy , Proto-Oncogene Proteins c-bcl-2/biosynthesis , Sulfonamides/pharmacology , Tumor Suppressor Protein p53/metabolism , Aged , Aged, 80 and over , Annexin A5/metabolism , Caspase 3/metabolism , Cytochromes c/metabolism , Cytosol/metabolism , Cytosol/pathology , Dose-Response Relationship, Drug , Drug Resistance/drug effects , Drug Screening Assays, Antitumor , Female , Hematopoietic Stem Cells/metabolism , Hematopoietic Stem Cells/pathology , Humans , Leukemia, Lymphocytic, Chronic, B-Cell/metabolism , Leukemia, Lymphocytic, Chronic, B-Cell/pathology , Male , Middle Aged , Mitochondria/metabolism , Mitochondria/pathology , Poly(ADP-ribose) Polymerases/metabolism , Protein Transport/drug effects , T-Lymphocytes/metabolism , T-Lymphocytes/pathology , Time Factors , Tumor Cells, Cultured , Up-Regulation/drug effects , bcl-2-Associated X Protein/metabolismABSTRACT
The alpha-helical amphipathic peptide D-(KLAKLAK)2 is toxic to eukaryotic cells if internalized by a suitable targeting mechanism. We have targeted this peptide to malignant hemopoietic cells via conjugation to monoclonal antibodies, which recognize lineage-specific cell surface molecules. An anti-CD19/peptide conjugate efficiently killed 3/3 B lymphoid lines. However, an anti-CD33/peptide conjugate was cytotoxic to only one of three CD33-positive myeloid leukemia lines. The IC50 towards susceptible lines were in the low nanomolar range. Conjugates were highly selective and did not kill cells that did not express the appropriate cell surface cognate of the antibody moiety. Anti-CD19/peptide conjugates efficiently killed cells from patients with chronic lymphocytic leukemia but anti-CD33/peptide reagents were less effective against fresh acute myeloid leukemia cells. We therefore suggest that amphipathic peptides may be of value as targeted therapeutic agents for the treatment of a subset of hematologic malignancies.
Subject(s)
Apoptosis/drug effects , Hematologic Neoplasms/drug therapy , Immunotoxins/pharmacology , Peptides/administration & dosage , Acute Disease , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/metabolism , Antigens, CD/immunology , Antigens, CD19/immunology , Antigens, Differentiation, Myelomonocytic/immunology , B-Lymphocytes/drug effects , Cell Line, Tumor , Drug Screening Assays, Antitumor , Hematologic Neoplasms/immunology , Humans , Leukemia, Lymphocytic, Chronic, B-Cell/drug therapy , Leukemia, Lymphocytic, Chronic, B-Cell/immunology , Leukemia, Myeloid/drug therapy , Leukemia, Myeloid/immunology , Lymphoma/drug therapy , Lymphoma/immunology , Sialic Acid Binding Ig-like Lectin 3ABSTRACT
We studied the actions of geldanamycin (GA) and herbimycin A (HMA), inhibitors of the chaperone proteins Hsp90 and GRP94, on B chronic lymphocytic leukemia (CLL) cells in vitro. Both drugs induced apoptosis of the majority of CLL isolates studied. Whereas exposure to 4-hour pulses of 30 to 100 nM GA killed normal B lymphocytes and CLL cells with similar dose responses, T lymphocytes from healthy donors as well as those present in the CLL isolates were relatively resistant. GA, but not HMA, showed a modest cytoprotective effect toward CD34+ hematopoietic progenitors from normal bone marrow. The ability of bone marrow progenitors to form hematopoietic colonies was unaffected by pulse exposures to GA. Both GA and HMA synergized with chlorambucil and fludarabine in killing a subset of CLL isolates. GA- and HMA-induced apoptosis was preceded by the up-regulation of the stress-responsive chaperones Hsp70 and BiP. Both ansamycins also resulted in down-regulation of Akt protein kinase, a modulator of cell survival. The relative resistance of T lymphocytes and of CD34+ bone marrow progenitors to GA coupled with its ability to induce apoptosis following brief exposures and to synergize with cytotoxic drugs warrant further investigation of ansamycins as potential therapeutic agents in CLL.
