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Poly (ADP-ribose) polymerase cleavage monitored in situ in apoptotic cells
M A O'Brien1, R A Moravec, T L Riss
1Promega Corporation, Madison, WI, USA. mobrien@promega.com
Abstract:
During apoptosis, the activation of a family of cysteine proteases, or caspases, results in proteolytic cleavage of numerous substrates. Antibody probes specific for neoepitopes on protein fragments generated by caspase cleavage provide a means to monitor caspase activity at the level of the individual cell. Poly (ADP-ribose) polymerase (PARP), a nuclear enzyme involved in DNA repair, is a well-known substrate for caspase-3 cleavage during apoptosis. Its cleavage is considered to be a hallmark of apoptosis. Here, we demonstrate that an affinity-purified polyclonal antibody to the p85 fragment of PARP is specific for apoptotic cells. Western blots show that the antibody recognizes the 85-kDa (p85) fragment of PARP but not full-length PARP. We demonstrate a time course of PARP cleavage and DNA fragmentation in situ using the PARP p85 fragment antibody and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) in Jurkat cells treated with anti-Fas. Furthermore, our results indicate that the p85 fragment of PARP resulting from caspase cleavage during apoptosis is rapidly localized outside the condensed chromatin but not in the cytoplasm.
Insights
This study introduces a new antibody that specifically detects the p85 fragment of Poly (ADP-ribose) polymerase (PARP) in apoptotic cells. This tool aids in monitoring caspase activity and understanding apoptosis progression.
Area of Science:
- Cell Biology
- Biochemistry
- Molecular Biology
Background:
- Apoptosis involves caspase activation, leading to substrate cleavage.
- Poly (ADP-ribose) polymerase (PARP) cleavage by caspase-3 is a hallmark of apoptosis.
- Monitoring caspase activity at the single-cell level requires specific probes.
Purpose of the Study:
- To develop and validate an antibody specific for the caspase-cleaved p85 fragment of PARP.
- To assess the utility of this antibody for detecting apoptotic cells.
- To investigate the kinetics of PARP cleavage and its subcellular localization during apoptosis.
Main Methods:
- Western blotting to confirm antibody specificity for the PARP p85 fragment.
- In situ detection of PARP cleavage and DNA fragmentation using the PARP p85 antibody and TUNEL assay.
- Time-course analysis in Jurkat cells treated with anti-Fas.
Main Results:
- An affinity-purified polyclonal antibody specifically recognizes the 85-kDa (p85) fragment of PARP, not full-length PARP.
- The antibody successfully detects PARP cleavage and DNA fragmentation in apoptotic Jurkat cells.
- The PARP p85 fragment localizes outside condensed chromatin but not in the cytoplasm during apoptosis.
Conclusions:
- The anti-PARP p85 fragment antibody is a specific marker for apoptotic cells.
- This antibody enables real-time monitoring of caspase activity and apoptosis.
- The localization of the PARP p85 fragment provides insights into apoptotic mechanisms.