An In Vitro Assay for the Quantitation of PARP Activity in Cell Extracts and for Determining the Efficacy of PARP Inhibitors

Poly(ADP-ribose) polymerase-1 (PARP-1) is a nuclear enzyme activated by DNA breaks. Activated PARP-1 cleaves NAD into nicotinamide and ADP-ribose and catalyzes the transfer of ADP-ribose units from NAD+ to target nuclear proteins. Poly(ADP-ribosyl)ation of proteins has been implicated in the regulation of a diverse array of cellular processes ranging from DNA repair⁽¹⁾ and genetic stability⁽²⁾ to chromatin organization,⁽³⁾ transcription,⁽⁴⁾ replication⁽⁵⁾ and protein degradation.⁽⁶⁾ Moderate activation of PARP-1 facilitates the efficient repair of DNA damage. However, overactivation of PARP has been implicated in the pathogenesis of several diseases, including stroke,⁽⁷⁾ myocardial infarction,⁽⁸⁾ diabetes,⁽⁹⁾ shock,⁽¹⁰⁾ neurodegenerative disorders⁽¹¹⁾ and allergy.⁽¹²⁾ PARP inhibitors show promise in improving cardiac and vascular dysfunction associated with advanced aging,⁽¹³⁾ preventing allergen-induced asthma-like reactions in sensitized Guinea pigs,⁽¹⁴⁾ and in augmenting the activity of Topoisomerase inhibitors in the treatment of cancer.⁽¹⁵⁾

To facilitate PARP research, we have developed a simple and rapid assay for the in vitro quantitation of PARP in cell extracts and for screening of PARP inhibitors in a 96-well format. The assay measures the poly(ADP-ribosylation) of histone proteins immobilized in the wells of a 96-well plate using biotinylated NAD as substrate. We investigated the assay for its ability to measure changes in PARP activity in WEHI cells induced to undergo apoptosis with etoposide, and for measurement of IC₅₀ values of three popular PARP inhibitors, 3-aminobenzamide, benzamide, and 4-amino-1,8-naphthalimide (all Trevigen, Inc., Gaithersburg, MD).

Reagent-grade chemicals were obtained from Sigma (St. Louis, MO). WEHI cells (ATCC, Manassas, VA), derived from a murine T cell leukemia, were grown in RPMI 1640 medium (Invitrogen, Carlsbad, CA) containing 10% fetal bovine serum (HyClone, Logan, UT) at 37 C in an atmosphere of 5% CO₂. The cells were suspended at a density of 0.5 × 10⁶/ml in RPMI 1640 medium containing 10% fetal bovine serum. Etoposide was added to a final concentration of 50 μM to induce apoptosis. The cells were incubated at 37 C and 5% CO₂ for various times up to 6 hours and harvested. The cells were washed once with 1× PBS and lysed with a buffer containing 1× PARP Assay Buffer, 400 mM NaCl, 1% (v/v) Triton X-100, and 0.4 mM Phenylmethylsulfonyl fluoride. The lysates were centrifuged at 10,000 rpm for 10 minutes at 4 C. The supernatants were recovered and assayed for protein concentration by the Bradford procedure.⁽¹⁶⁾ Clarified extracts were stored at 㫨 C until assayed.

Immediately prior to performing the assay, the following reagents were prepared: A) 20× PARP Assay Buffer (Trevigen) was diluted to 1× with dH₂O; B) 10× PARP Cocktail (Trevigen), which contains a mixture of NAD and biotinylated NAD, was diluted to 1× by the addition of Activated DNA (Trevigen) and 1× PARP Assay Buffer; C) serial dilutions of PARP enzyme (Trevigen) and the PARP inhibitors were prepared with 1× PARP Assay Buffer; D) 10× Strep-Diluent (Trevigen) was diluted to 1× with 1× PBS.

The colorimetric and chemiluminescent assays for PARP activity were performed in clear (Trevigen) or white (Trevigen) 96-well plates, respectively, precoated with histones. First, 25 μl of the diluted PARP Cocktail was added to all the wells. Serial dilutions of PARP enzyme were added to triplicate wells to generate the standard curve. To generate inhibitor curves, serial dilutions of the PARP inhibitors were added to triplicate wells containing either 1 unit of PARP/well for the colorimetric readout, or 0.1 unit of PARP/well for the chemiluminescent readout. Clarified cell extracts (10 μl/well) were added to triplicate wells for determining cellular PARP activity. Controls included background wells with 1× Assay Buffer alone (no PARP) and wells with no inhibitor for determining the maximum or 100% PARP activity value. In all cases the final reaction volume was 50 μl. The reactions were allowed to proceed for 1 hour at room temperature. The plate was washed 4 times with 1× PBS and then incubated for 20 minutes with 50 μl/well Strep-HRP (Trevigen), diluted 1:500 with 1× Strep-Diluent (Trevigen). The plate was washed 4 times with 1× PBS in preparation for the addition of the HRP substrate.

For the colorimetric readout, 50 μl of TACS-Sapphire (Trevigen) was added to each well and incubated in the dark at room temperature for 15 minutes. TACS-Sapphire is an HRP substrate generating a soluble blue color with maximum absorbance at 630 nm. Development of the colorimetric reaction can be stopped by the addition of an equal volume of 0.2 M HCl or 5% phosphoric acid, generating a yellow color stable for up to 60 minutes that can be read at 450 nm. The absorbance at 450 nm will be significantly higher than the absorbance at 630 nm.

For the chemiluminescence readout, a two-component substrate (Trevigen) was mixed together in equal proportions and 100 μl was added per well. The light output was immediately determined in a plate reader set up for measuring chemiluminescence (BMG Laboratories Fluostar Optima, Durham, NC).

A typical colorimetric assay readout has the appearance shown in Figure 1. The TACS-Sapphire substrate yields a blue color in the presence of HRP, which dynamically increases in intensity with time and allows scientists with the appropriate plate reader to measure the change in absorbance at 630 nm with time. The addition of 0.2 M HCl or 5% phosphoric acid stops the reaction and changes the color to yellow with an absorption maximum at 450 nm.

The colorimetric curves for the PARP activity standard curve and the inhibition curves for the PARP inhibitors are graphically represented in Figure 2. In the presence of 1 unit of PARP and 68 μM total NAD, the IC₅₀ values for 3-aminobenzamide, benzamide, and 4-amino-1,8-naphthalimide were 20.0 μM, 1.6 μM, and 0.2 μM, respectively. These values compare favorably to published IC₅₀ values of 5.4 μM⁽¹⁷⁾ or 33 μM⁽¹⁸⁾ for 3-aminobenzamide, 3.3 μM⁽¹⁷⁾ for benzamide, and 0.18 μM for 4-amino-1,8-naphthalimide.⁽¹⁸⁾

     The chemiluminescent curves for the PARP activity standard curve and the inhibition curves for the PARP inhibitors are graphically represented in Figure 3. In the presence of 0.1 unit of PARP and 68 μM total NAD, the IC₅₀ values for 3-aminobenzamide, benzamide, and 4-amino-1,8-naphthalimide were 5.0 μM, 0.32 μM, and 0.05 μM, respectively. The chemiluminescent assay exhibited a broader dynamic range and approximately a 10-fold increase in sensitivity than the colorimetric assay format.

PARP is constitutively expressed at high levels in a variety of tissues and acts to monitor the state of health of the cells. Upon DNA damage, the PARP-1 DNA binding domain binds specifically to DNA single and double strand breaks. This binding induces a conformational change in the PARP protein, activates the catalytic activity of the enzyme, and leads to an increase in poly(ADP-ribose) synthesis on target DNA binding proteins by more than 100-fold. The assay was applied to measure changes in endogenous activity of PARP in WEHI cells exposed to 50 μM etoposide. The results are shown in Figure 4. PARP activity in WEHI cells exposed to 50 μM etoposide increased to a maximum level within 1 hour of exposure and then decreased below levels in the cells not exposed to the drug. The high endogenous level of PARP in the untreated WEHI cells results from sampling the cells initially at too high a cell density. Under these conditions, WEHI cells exhibit elevated endogenous levels of nuclear poly(ADP-ribosylation) and the appearance of small comets in a single-cell gel electrophoresis comet assay (unpublished observations).⁽¹⁹⁾

In summary, we have developed a simple, rapid and sensitive assay for the quantitation of PARP activity in cell extracts and for determining the efficacy of PARP inhibitors. The assay yielded IC₅₀ values for 3-aminobenzamide, benzamide, and 4-amino-1,8-naphthalimide which correlated with those found in the literature. Finally, the assay detected transient changes in PARP activity in WEHI cells exposed to etoposide. The colorimetric and chemiluminescent readout of the assay obviates the need for radioactive NAD substrates and should facilitate the search for potent PARP inhibitors for the treatment of a variety of diseases including neurodegenerative disorders⁽²⁰⁾ and stroke.⁽²¹⁾

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