Programmed Cell Death-Assaying PARP Activity

There are various ways for cells to die, each having its own implication for the health of the organism. Among these are several apoptotic (programmed cell death) pathways that are invoked in response to specific intrinsic or extrinsic signals. These pathways lead to controlled, orderly series of distinct biochemical and morphological changes that typically result in the cell being phagocytosed, leaving behind no traces that would otherwise lead to inflammation.

Scientists distinguish apoptotic cells by monitoring and measuring biochemical or morphological changes that take place in the cell. Damaged DNA is not the same as degraded DNA, although both can lead to cell death. Understanding the cause and effects enables researchers to characterize a potential drug’s mode of action, for example, or search for inhibitors of different steps in a pathway as they relate to cell health. Previous Biocompare articles have examined assays for caspases, a family of proteases intricately involved with apoptosis, and TUNEL assays, which detect the nicked DNA that is characteristic of late-stage apoptosis. One of the substrates of certain caspases is an enzyme involved in detection of nicked DNA, poly(ADP-ribose) polymerase 1 (PARP). Here we look at ways to examine PARP in the context of apoptosis.

What is PARP?

PARP is a 113-kD nuclear enzyme that is activated by single-strand DNA breaks. It catalyzes the sequential addition of ADP ribose subunits from nicotinamide adenine dinucleotide (NAD(+)) onto protein acceptors, including histones, some transcription factors and even PARP itself. “As soon as the cell detects the DNA is damaged, PARP activity increases 500-fold in a few minutes. Then the NAD level drops about 5-fold within about 15 minutes. People want to see that when they’re looking at a new drug or chemotherapy agent—they want to see whether there’s damage to the cells, and they want to see what happens to PARP activity,” says Chandra Mohan, EMD Millipore’s senior manager, technical writing and documentation development.

Poly(ADP-ribose)ylation (PARylation) leads to a recruitment of DNA repair factors and can also contribute to an opening up of the chromosome, giving those factors better access to the damaged site. Too much DNA damage may induce apoptosis, during which PARP is cleaved into fragments of approximately 89 and 24 kD, inactivating it. Even more extensive DNA breakage (as might be seen following massive oxidative stress, for example) can kick PARP activity into overdrive, leading to a rapid depletion of NAD(+), which in turn can engender energy depletion and necrosis [1].

When PARP activity is blocked, single-strand DNA break repair is inhibited. Ordinarily, this defect is corrected for during replication when the break is converted into a double-strand break and then repaired through the homologous recombination pathway. But in some diseases—including many breast cancers with mutations in BRCA1 or BRCA2, for example—this pathway is also defective, so the two defects exist in what scientists at Trevigen call a “synthetic lethal relationship”: Neither mutation itself is lethal, but mutations in both pathways will cause the cells to die.

Is PARP active?

Researchers may look at PARP activity to screen for inhibitors (or activators), notes Franka Maurer, applications specialist with BMG LABTECH GmbH.

Tools for this purpose may, like Trevigen’s Universal PARP Assay Kit, assay for the ability of samples to catalyze the incorporation of biotinylated NAD into PAR and onto immobilized histones, a reaction that can be quantified by chemiluminescence or colorimetrically in a microplate reader. Trevigen, AMSBio and others offer related PARP-activity assays, as well, including some that measure the levels of PAR found in samples and (inhibition of) consumption of NAD.

Many similar assays also can be performed by immunohistochemistry (IHC), flow cytometry or Western blotting.

Is PARP there and intact?

There are many commercially available antibodies for PARP detection as well as a variety of assay kits, including for ELISAs, Westerns, and IHC formats.

ChromoTek GmbH offers a Chromobody® plasmid for PARP: Upon transfection, cells express an extremely small functional anti-PARP antibody fused to a chromophore, which can be used for intracellular imaging, explains marketing and sales manager Christoph Eckert.

Yet when it comes to examining its role in apoptosis, the presence or cellular location of PARP may not be as revealing as whether the molecule is full-length or cleaved (or which cleavage products are found). During apoptosis, human PARP is cleaved between aspartic acid 214 and glycine 215 residues. Antibodies can be found that will recognize both intact PARP and the 89-kD fragment of cleaved PARP—which is useful for techniques such as Western blotting.

Antibodies also have been developed that will specifically recognize the 214/215 cleavage site (and not intact protein), and many of these have been incorporated into ELISA and Luminex-based kits, says James Deng, product manager for ELISAs at Thermo Fisher Scientific. Such products include an ELISA kit from Thermo Scientific Pierce that measures cleaved PARP in whole cells using a near-infrared (NIR) fluorophore.

Necrotic mechanisms are not as tidy. “When you do a Western and see multiple [PARP] fragments, chances are you’ll say this is not apoptosis but more likely necrosis,” says Mohan. “Or if you see bands at 89, 24, 50 and 35 kD, that means there is a combination of processes, in which some cells died of necrosis and some of apoptosis.”

Multiple measures

When monitoring cell death, researchers often examine more than one measure—to see which pathway is being activated, for example, or at what stage some phenomenon is occurring relative to treatment. Multiple different assays can be performed—looking for membrane blebbing, chromatin condensation and the externalization of phosphatidylserine on the plasma membrane, which are characteristic of apoptotic cell death—or a single assay type can be performed in multiplex.

Both EMD Millipore and Thermo Fisher Scientific offer Luminex-based kits for PARP detection, which are available as multiplex kits or can be multiplexed with assays for other analytes. BD Biosciences offers a multiplex apoptosis kit for flow cytometry that includes detection of cleaved PARP. These types of multiplex assays, notes Mohan, are especially useful when there is only a limited amount of sample.

Whether you’re trying to differentiate apoptosis from necrosis or autophagy, or asking whether a compound is an effective PARP inhibitor or activator, it may be easier to find an appropriate tool than to decide which of many available tools to use.

Reference

[1] Horvath, EM, Zsengellér, ZK, Szabo, C, “Quantification of PARP activity in human tissues: ex vivo assays in blood cells and immunohistochemistry in human biopsies,” Methods Mol Biol, 780:267-75, 2011. [PMID: 21870266]