Deficient activation of Bak and Bax confers resistance to gemtuzumab ozogamicin-induced apoptotic cell death in AML.
OBJECTIVE
Gemtuzumab ozogamicin (GO), comprising a CD33 antibody linked to the toxin calicheamicin, represents a novel and promising targeted therapy in acute myeloid leukemia (AML). The more definite mechanisms by which GO exerts its cell death-inducing propensity, and thus how sensitivity and resistance to GO are regulated, still remain to be elucidated. We have studied proapoptotic signaling events induced by GO and free calicheamicin in AML cells.


METHODS
AML cell lines and primary blood cells from six patients with acute leukemia were incubated with GO or calicheamicin and the effects on cell viability and proapoptotic signaling were analyzed using MTT assay, flow cytometry, immunofluorescence and immunoblotting.


RESULTS
GO and free calicheamicin at clinically relevant concentrations resulted in decreased cell viability, appearance of apoptotic morphology, depolarization of mitochondria, and activation of caspase-3 signaling in HL60 and NB4 AML cells. In contrast, none of these events were observed in GO-exposed KG1a AML cells. Notably, GO treatment also caused proapoptotic conformation of Bak and Bax and activation of stress-activated protein kinase p38 in responsive but not in resistant AML cells. In patient-derived AML cells, GO and calicheamicin induced a heterogeneous cytotoxic response, partly linked to CD33 expression and with signs of caspase-3 activation.


CONCLUSIONS
Our novel data on GO-induced proapoptotic activation of Bax, Bak, and stress-activated protein kinase indicate an important role for these signal proteins in the regulation of GO sensitivity in AML.
Haag
Petra
P
Karolinska Biomics Center, Department of Oncology/Pathology, Stockholm, Sweden.
Viktorsson
Kristina
K
Lindberg
Marita Lagergren
ML
Kanter
Lena
L
Lewensohn
Rolf
R
Stenke
Leif
L
D000617:Aminoglycosides / Q000494:pharmacology*; D000911:Antibodies, Monoclonal / Q000494:pharmacology*; D061067:Antibodies, Monoclonal, Humanized; D017209:Apoptosis / Q000187:drug effects*; D053148:Caspase 3 / Q000378:metabolism; D045744:Cell Line, Tumor; D004351:Drug Resistance; D000079982:Gemtuzumab; D006801:Humans; D015470:Leukemia, Myeloid, Acute / Q000188:drug therapy / Q000473:pathology*; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D048055:Mitogen-Activated Protein Kinase 8 / Q000378:metabolism; D014407:Tumor Cells, Cultured; D050998:bcl-2 Homologous Antagonist-Killer Protein / Q000378:metabolism / Q000502:physiology*; D051028:bcl-2-Associated X Protein / Q000378:metabolism / Q000502:physiology*
D000617:Aminoglycosides; D000911:Antibodies, Monoclonal; D061067:Antibodies, Monoclonal, Humanized; C494772:BAK1 protein, human; D050998:bcl-2 Homologous Antagonist-Killer Protein; D051028:bcl-2-Associated X Protein; D000079982:Gemtuzumab; D048055:Mitogen-Activated Protein Kinase 8; D053148:Caspase 3