Oligonucleosomal DNA fragmentation in MCF-7 cells undergoing palmitate-induced apoptosis.
Oligonucleosomal fragmentation of nuclear DNA is the late-stage apoptosis hallmark. In apoptotic mammalian cells the fragmentation is catalyzed by DFF40/CAD DNase primarily activated by caspase 3 through the site-specific proteolytic cleavage of DFF45/ICAD. A deletion in the casp3 gene of human breast adenocarcinoma MCF-7 results in lack of procaspase 3 in these cells. The absence of caspase 3 in MCF-7 leads to disability to activate oligonucleosomal DNA fragmentation in TNF-alpha induced cell death. In this study, sodium palmitate was used as an apoptotic stimulus for MCF-7. It has been shown that palmitate but not TNF-alpha induces both apoptotic changes in nuclei and oligonucleosomal DNA fragmentation in casp3-mutated MCF-7. Activation and accumulation of 40-50 kD DFF40-like DNases in nuclei of palmitate-treated apoptotic MCF-7 were detected by SDS-DNA-PAGE assay. Microsomal fraction of apoptotic MCF-7 does not contain any detectable DNases, but activates 40-50 kD nucleases when incubated with human placental chromatin. Furthermore, microsomes of apoptotic MCF-7 induce oligonucleosomal fragmentation of chromatin in a cell-free system. Both the activation of DNases and chromatin fragmentation are suppressed in the presence of the caspase 3/7 inhibitor Ac-DEVD-CHO. Microsome-associated caspase 7 is suggested to play an essential role in the induction of oligonucleosomal DNA fragmentation in casp3-deficient MCF-7 cells.
Semenov
D V
DV
Novosibirsk Institute of Bioorganic Chemistry, Siberian Division of the Russian Academy of Sciences, Novosibirsk 630090, Russia. semenov@niboch.nsc.ru
Aronov
P A
PA
Kuligina
E V
EV
Potapenko
M O
MO
Richter
V A
VA
D017209:Apoptosis / Q000187:drug effects*; D053148:Caspase 3; D020169:Caspases / Q000235:genetics; D045744:Cell Line, Tumor; D053938:DNA Fragmentation / Q000187:drug effects*; D004273:DNA, Neoplasm / Q000378:metabolism*; D003851:Deoxyribonucleases / Q000378:metabolism; D005091:Exons / Q000235:genetics; D006801:Humans; D008856:Microscopy, Fluorescence; D009707:Nucleosomes / Q000737:chemistry / Q000187:drug effects* / Q000378:metabolism*; D010168:Palmitates / Q000494:pharmacology*; D010920:Placenta / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D017384:Sequence Deletion / Q000235:genetics; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D004273:DNA, Neoplasm; D009707:Nucleosomes; D010168:Palmitates; D014409:Tumor Necrosis Factor-alpha; D003851:Deoxyribonucleases; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases