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Oligonucleosomal DNA fragmentation in MCF-7 cells undergoing palmitate-induced apoptosis

D V Semenov1, P A Aronov, E V Kuligina

  • 1Novosibirsk Institute of Bioorganic Chemistry, Siberian Division of the Russian Academy of Sciences, Novosibirsk 630090, Russia. semenov@niboch.nsc.ru

Biochemistry. Biokhimiia
|February 6, 2004
PubMed

Insights

Sodium palmitate induces DNA fragmentation in caspase-3-deficient cells, activating DFF40-like nucleases. Microsomes containing caspase-7 play a key role in this process during apoptosis.

Area of Science:

  • Cell Biology
  • Molecular Biology
  • Biochemistry

Background:

  • Oligonucleosomal DNA fragmentation is a hallmark of late-stage apoptosis, typically mediated by DFF40/CAD DNase.
  • DFF40/CAD activation relies on caspase 3-mediated cleavage of its inhibitor, DFF45/ICAD.
  • Human breast adenocarcinoma MCF-7 cells with a casp3 gene deletion lack functional caspase 3, impairing TNF-alpha-induced DNA fragmentation.

Purpose of the Study:

  • To investigate the mechanism of DNA fragmentation in caspase-3-deficient MCF-7 cells induced by sodium palmitate.
  • To identify the nucleases involved and their activation pathways in the absence of caspase 3.

Main Methods:

  • Induction of apoptosis in casp3-mutated MCF-7 cells using sodium palmitate.
  • Analysis of nuclear DNA fragmentation via SDS-DNA-PAGE assay.
  • Investigation of nuclease activity in microsomal fractions using human placental chromatin in a cell-free system.
  • Assessment of caspase inhibition using Ac-DEVD-CHO.

Main Results:

  • Sodium palmitate, unlike TNF-alpha, induced apoptotic changes and oligonucleosomal DNA fragmentation in casp3-deficient MCF-7 cells.
  • Activation and nuclear accumulation of 40-50 kD DFF40-like DNases were observed following palmitate treatment.
  • Microsomal fractions from apoptotic MCF-7 cells activated nucleases and fragmented chromatin in a cell-free system, an effect suppressed by a caspase 3/7 inhibitor.

Conclusions:

  • Microsome-associated caspase 7 is implicated in the activation of DFF40-like nucleases and subsequent DNA fragmentation in caspase-3-deficient cells.
  • This study reveals an alternative pathway for DNA fragmentation during apoptosis independent of the canonical caspase-3-mediated activation of DFF40/CAD.

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