PARP-1^+/+ and PARP-1^-/- 3T3s were exposed to increasing concentrations of NCS and cytotoxicity was determined through a growth assay (see Methods). Below 1.5 nM NCS PARP-1^+/+ and PARP-1^-/- 3T3s demonstrated exactly the same susceptibility to the lethal effect of NCS (Figure 1A). However, PARP-1^+/+ were substantially more sensitive to NCS than PARP-1^-/- 3T3s in the high range of NCS concentration.

Pre-treatment with the PARP-1 inhibitor 4-amino-1,8-naphthalimide (ANI) did not alter the susceptibility of PARP-1^+/+ 3T3s to NCS (Figure 2A). In contrast, PARP-1^-/- were considerably more sensitive than PARP-1^+/+ 3T3s to the lethal effect of γ-rays (Figure 2B) and H₂O₂ (Figure 2C).

The PARP-1^+/+ and PARP-1^-/- 3T3 clones used in these experiments originated each from independent isolates immortalized at random. There was therefore a possibility that minor differences might exist between both cell lines with regard to cell death mechanism or efficiency at a high level of DSB, and contribute to altered NCS susceptibility in addition to the PARP-1 defect. To settle this question, we used PARP-1^-/- 3T3s in which the full PARP-1 cDNA had been re-inserted by retroviral infection with a pBabe construct, allowing complete restoration of the PARP-1 activity 38. The NCS response of these cells was compared to that of PARP-1^-/- 3T3s from the same clonal isolate transfected with the void vector. Both cell lines showed exactly the same susceptibility to NCS, with a purely exponential dose-dependence (Figure 1B).

DSB in PARP-1^+/+ and PARP-1^-/- 3T3s were measured by PFGE. Briefly, cells were exposed to increasing concentrations of NCS, up to 30 nM for 10-min at 37°C, then chilled in ice, harvested in PBS-EDTA buffer, and inserted into agarose plugs and lysed for PFGE analysis. The effect of NCS was compared to that of γ-rays (up to 60 Gy) on cells irradiated in ice.

The incidence of DSB was found to increase linearly with the γ-ray dose, in agreement with earlier reports 39. It also grew linearly with the NCS concentration, and incubation with 1 nM NCS yielded the same amount of DSB as 1.21 Gy γ-rays (data not shown). PARP-1^+/+ and PARP-1^-/- 3T3s did not show any difference in this assay. Taking into account the fact that 1 Gy γ-rays produces 41 DSB per diploid cell nucleus 40, the average incidence of DSB formed by 1 nM NCS in each 3T3 fibroblast was estimated at ca. 50 DSB. With consideration to the number of DSB induced by the treatment, the lethal efficiency of NCS was in the same range as that for γ-rays (Table 1).

To determine whether the repair of NCS-induced DSB was deficient in PARP-1^-/- relative to PARP-1^+/+ 3T3s, DSB rejoining in cells treated with 30 nM NCS was measured through PFGE. The data (Figure 3) did not demonstrate any difference in the rejoining kinetics between both cell lines.

PARP-1^+/+ and PARP-1^-/- 3T3s were assayed through immunoflurescence for the determination of (i) PARP-1 expression, (ii) PARP-1 activity visualized by pADPr synthesis following γ-ray irradiation, NCS and H₂O₂, and (iii) ATM-dependent H2AX histone phosphorylation in response to DNA damage induced by γ-rays, H₂O₂ or NCS. The results are shown in Figure 4.

PARP-1 expression was detected in the nucleus of PARP-1^+/+ 3T3s and was missing in PARP-1^-/- 3T3s, as expected. Also as expected, pADPr synthesis was observed selectively in the nucleus of PARP-1^+/+ cells following γ-ray or H₂O₂ exposure, and was repressed by ANI. Stable transfection with a retroviral vector coding for the full PARP-1 gene, restored PARP-1 expression and function.

In contrast to γ-rays or H₂O₂, NCS did not induce any measurable pADPr synthesis in PARP-1^+/+ 3T3s, even at a concentration of 30 nM representing ca. 20-fold the IC₅₀ value and yielding the same amount of DSB as 36 Gy radiation. However, consistent with induction of DSB in chromosomal DNA, NCS at relatively low concentration (2 nM) initiated rapid phosphorylation of H2AX in the same way as γ-rays or H₂O₂. In agreement with this observation, MRE11 phosphorylation and focus formation through an ATM- and NBS1-dependent mechanism, has recently been shown to occur after NCS treatment 37.

Mouse monoclonal antibodies directed against the PARP-1 C-terminal domain (clone 7-D3-6) and pADPr (clone 10 H) were from Becton-Dickinson Biosciences and Alexis Corporation, respectively. Rabbit polyclonal antibody raised against γ-H2AX, was from Trevigen. Alexa-488^®-conjugated goat anti-rabbit and anti-mouse secondary antibodies, were purchased from Molecular Probes. HRP-conjugated goat anti-mouse and anti-rabbit IgG came from Jackson Immunoresearch.

[2-¹⁴C]Thymidine was purchased from PerkinElmer Life Sciences. 4-amino-1,8-naphthalimide (ANI) came from ACROS Organics. H₂O₂ was from Sigma-Aldrich. Other chemicals were of the highest purity available and came from VWR International. The products for cell culture were from Invitrogen.

The neocarzinostatin holoprotein, prepared and titrated as described 60, was stored as a sterile 1 mM stock solution in 2 mM sodium formate buffer, pH 4.0, at liquid nitrogen temperature.

Spontaneously immortalized 3T3 mouse embryo fibroblasts obtained from homozygous wild-type (PARP-1^+/+) and knockout (PARP-1^-/-) C57BL/6 mice 44, were provided by Dr. Gilbert de Murcia. It should be stressed that the knockout in PARP-1^-/- fibroblasts was complete, i. e., no fragment of the functional domain of PARP-1 protein was expressed in these cells. PARP-1^-/- 3T3s in which the full PARP-1 gene was reinserted by a retroviral pBabe-puro construct 38 were a generous gift of Dr. Moshe Oren.

3T3 fibroblasts were maintained as exponentially growing monolayers in Dulbecco modified Eagle's minimum essential medium (DMEM), with antibiotics and 10% foetal calf serum as described 6. The mean doubling time of cells was 27-h and 52-h for PARP-1^+/+ and PARP-1^-/- 3T3s, respectively.

Due to a low cloning efficiency of 3T3 fibroblasts in a feeder layer technique, all cytotoxicity determinations were performed using growth assays. PARP-1^+/+ or PARP-1^-/- 3T3 fibroblasts were plated in triplicate (10⁵ cells per 25 cm² vented flasks) and incubated overnight before treatment. After treatment, the flasks were rinsed twice with HBSS and returned to fresh medium for exactly 5 doubling times, with one medium change at day 7 for PARP-1^-/- 3T3s. Fibroblasts were harvested by 0.05% trypsin-0.02% EDTA, pelleted, resuspended in medium and counted under microscope in a Malassez cuvette.

Irradiation of cells was performed at room temperature in medium equilibrated with 5% CO₂ in air, using an IBL-637 (¹³⁷Cs) γ-ray irradiator (Cis-Biointernational). The dose rate was 0.86 Gy/min or 8.0 Gy/min for survival or immunofluorescence studies, respectively.

Aliquots of NCS were thawed just before use, adjusted to the suitable dilution in ice-cold PBS buffer, pH 6.0, and immediately introduced into culture flasks (25 cm², 5 ml medium) with gentle agitation. The whole treatment was performed in dim light to avoid photo-induced degradation of the drug. The cytotoxic effect of NCS at fixed concentration was investigated as a function of the length of drug exposure. Cytotoxicity increased steeply with the length of contact and reached completion after 6-min only. More prolonged incubation did not result in increased cell death. Therefore, the length of contact with NCS was 10-min throughout.

For studies of H₂O₂ response, serial dilutions of concentrated (9.8 M) H₂O₂ were made in pure water, then in culture medium immediately prior to use. The length of contact with H₂O₂ was 10-min.

In some experiments ANI was used as a PARP-1 inhibitor 616263. When present, ANI (30 μM) from a 3 mM stock solution in pure DMSO was introduced 1-h prior to irradiation or NCS, and removed 1-h later with two HBSS washes. The DMSO concentration in medium was 1% and was kept constant throughout experiments with ANI. Controls were made with DMSO alone at the same concentration.

The determination of DSB formation and repair was carried out by pulsed field agarose gel electrophoresis (PFGE) through the clamped homogeneous electric field (CHEF) technique 6465. [2-¹⁴C]Thymidine-labeled cells (2–5 10⁶ cells per 25 cm² flask) were put in ice following treatment, harvested in ice-cold PBS supplemented with 2 mM EDTA by gentle scraping, and collected by sedimentation at 4°C. Pellets from 5 10⁵ cells were resuspended in 150 μl, final volume, of PBS buffer at 37°C, and mixed under mild vortexing with an equal volume of 1.6% w/v low-melting-point agarose (BDH Laboratory) in PBS buffer at 37°C. The suspension (300 μl) was immediately pipetted into pre-chilled 4 × 10 mm moulds and allowed to form plugs on ice for 30-min. Embedded cells were subsequently lysed by immersion of the plugs in 2 ml of a solution containing 2% N-lauroyl sarkosine, 40 mM EDTA, 1 mg/ml proteinase K in PBS, pH 7.8 and incubated, firstly for 2 h in ice, secondly for 24 h at 50°C. The plugs were subsequently washed twice with PBS, and treated for 1-h with 200 μg/ml RNase A. The plugs were finally washed twice with PBS and stored at 4°C overnight prior electrophoresis.

The plugs were inserted into the wells of an 0.8% w/v agarose gel (BioRad, chromosomal grade) made in 0.75 × TAE buffer (40 mM tris-acetate, 2 mM EDTA, pH 8), and the gels submitted to PFGE at 14°C in a CHEF-DR III apparatus (Bio-Rad) with buffer recirculation at 14°C. Migration was for 72 h at 2 V/cm with three switch times, namely, 1200-s, 1500-s and 1800-s with angles of 96°, 100° and 106°, respectively. The molecular weight markers were S. pombe and S. cerevisiae chromosomes (Bio-Rad).

After electrophoresis, the gels were stained with 0.5 μg/ml ethidium bromide under mild agitation (30-min), followed by destaining for 1-h in fresh buffer. DNA fluorescence was visualized over an UV transilluminator with camera recording. The gels were subsequently dried in vacuo over a piece of Whatman paper and analyzed using a Phosphorimager^® apparatus (Amersham Pharmacia Biotech), allowing a precise determination of the migration profile of DNA fragments and of the fractional radioactivity released from the plugs (FAR).

3T3 fibroblasts were grown for 24-h on coverslips and exposed to either γ-rays (5 Gy for γH2AX, 50 Gy for pADPR), H₂O₂ (1 mM, 10-min contact, 37°C), or graded concentrations of NCS (2 nM, 5 nM or 30 nM, 10-min contact, 37°C), and fixed 10-min after the beginning of treatment. For PARP-1 and pADPr immunofluorescence, fixation was by 4% formaldehyde (10-min, room temperature) in PBS followed by two PBS washes and neutralization of residual formaldehyde by 50 mM NH₄Cl (10-min, room temperature). After a further PBS wash (5-min, 4°C), cells were permeabilized by 0.5% Triton X-100 in PBS (5-min, 4°C), and finally rinsed twice with PBS. For γH2AX determination, cells were fixed in a 1:1 v:v acetone:methanol mixe (-20°C, 10-min), dried in air and rehydrated in PBS (15-min, room temperature).

Cell preparations on coverslips were subsequently incubated with 1% bovine serum albumin in PBS (10-min, room temperature) and exposed to anti-pADPr, anti-PARP-1 or anti-γH2AX primary antibody (1/200 dilution, 1-h, 37°C). The coverslips were rinsed thrice with PBS, and incubated with Alexa-488^®-conjugated secondary antibody (1/500 dilution, 30-min, 37°C), rinsed thrice with PBS, counterstained with 4,6-diamidino-2-phenylindole (DAPI) and mounted. Immunofluorescence was visualized using a Zeiss Axiophot microscope equipped with a Micromax chilled camera (Princeton Applied Research).