Induction of apoptotic DNA fragmentation and c-jun downregulation in human myeloid leukemia cells by the permeant Ca2+ chelator BAPTA/AM.
The permeant Ca2+ chelator acetoxymethyl-1,2-bis(2-aminopheoxy)ethane- N,N,N',N'-tetraacetic acid (BAPTA/AM), an agent previously used to characterize drug-induced apoptosis in neoplastic cells, has been examined with respect to induction of DNA fragmentation and cytotoxicity in the human leukemia cell lines HL-60 and U937. Exposure of cells to various concentrations of BAPTA/AM for 6 h resulted in a biphasic induction of internucleosomal DNA cleavage, with maximal damage occurring at 10-microM concentrations. Higher BAPTA/AM concentrations were associated with the loss of internucleosomal cleavage products, but with the appearance of larger (i.e., 50-kilobase) fragments on pulsed-field gel electrophoresis. Cells exposed to 10 microM BAPTA/AM exhibited classic apoptotic morphology, whereas cells exposed to 50-microM concentrations displayed atypical features (e.g., cell swelling, chromatin clumping); in each case, substantial cytotoxicity was noted. The actions of BAPTA/AM did not depend upon the presence of extracellular Ca2+, nor were they affected by impermeant Ca2+ chelators. Measurement of cytosolic Ca2+ by Fura-2/AM or Indo-1 revealed late but not early increases in intracellular Ca2+ in BAPTA/AM-treated cells. Finally, BAPTA/AM-induced apoptosis was accompanied by the concentration-dependent downregulation of the immediate early response gene c-jun. These findings suggest a complex role for Ca2+ chelators such as BAPTA/AM in the regulation of human myeloid leukemic cell apoptosis, and indicate that this agent may selectively antagonize internucleosomal DNA fragmentation without interfering with other aspects of the apoptotic response and/or cell lethality.
Grant
S
S
Department of Medicine, Medical College of Virginia, Richmond 23298, USA.
Freemerman
A J
AJ
Gregory
P C
PC
Martin
H A
HA
Turner
A J
AJ
Mikkelsen
R
R
Chelliah
J
J
Yanovich
S
S
Jarvis
W D
WD
D017209:Apoptosis / Q000187:drug effects; D002118:Calcium / Q000378:metabolism; D002614:Chelating Agents / Q000494:pharmacology; D004249:DNA Damage / Q000187:drug effects; D015536:Down-Regulation; D004533:Egtazic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology; D015973:Gene Expression Regulation, Leukemic / Q000235:genetics*; D016758:Genes, jun / Q000187:drug effects*; D006801:Humans; D007951:Leukemia, Myeloid / Q000235:genetics* / Q000378:metabolism / Q000473:pathology
D002614:Chelating Agents; C070379:1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester; D004533:Egtazic Acid; D002118:Calcium