Reagents were from Sigma-Aldrich (Sigma-Aldrich, St. Louis, MO, USA), unless otherwise specified. Plastic-wares were from Nunc (Nunc A/S, Roskilde, Denmark). A human colon adenocarcinoma cell line (LoVo WT), isolated from a metastatic nodule and its doxorubicin (DOX)-resistant variant (LoVo DX) were used in this study. Both cell lines were grown in monolayer in Ham’s F12 medium (Gibco BRL, Life Technologies, Paisley, UK) supplemented with 10% fetal bovine serum (FBS) (Hyclone, Cramlington NE23, UK), 1% L-glutamine (Gibco BRL, Life Technologies), 1% penicillin (50 U/ml)-streptomycin (50 μg/ml) (Gibco BRL, Life Technologies), 1% vitamins (Gibco BRL, Life Technologies) in a humidified atmosphere of 5% CO₂ in a water-jacketed incubator at 37°C. The pleiotropic drug-resistant cell line LoVo DX was obtained from its drug-sensitive parental LoVo cell line, by exposure to increasing concentrations of DOX (Adriblastina, Pharmacia and Upjohn, Milan, Italy) (24). Transfection and growth conditions of pcDNA3 transfected (NP), pcDNA3-SMOX transfected (NS) and mouse NB cell lines have been described (15,16). AA8, EM9 and UV61 cell lines (21,22) were a generous gift of Professor F. Palitti (Universita’ della Tuscia, Italy). Stable transfection with pcDNA3 and pcDNA3-SMOX were performed with Effectene (Qiagen) as described (16). Geneticin (G418) (300 mM) was the selection agent to isolate stable transfected pool of cells. X-irradiation of 1 and 10 cGy, were delivered by a Gilardoni CHF 320 G Unit (Gilardoni S.p.A., Mandello L., Italy) tested and complying with EU standards. Dose/rate was 0.1 Gy/min at 250 KeV, 1,5 A, with 0.5 mm Cu filter. Cells were irradiated on ice and fresh medium was replaced after exposure. Control cells were treated similarly, without irradiation. All experimental points were taken 6 and 24 h after irradiation.

To analyse cell proliferation we followed the instructions of the manufacturer of the Cell Proliferation Kit I (Roche). We performed triplicate experiments with increasing number of cell/plate (10³, 2×10³, 3×10³ cell/plate) normalised to 10³. Cells were seeded the day before the experiments and treated with the MTT solution for 2 h at growth conditions. Isopropanol (0.1 ml) with 0.04 N HCl was added to each well to quench the red phenol colour and the absorbance was measured on an ELISA plate reader with a test wavelength of 570 nm and a reference wavelength of 630 nm.

Level of intracellular hydrogen peroxide was determined by flow cytometry (FCM) analysis of fluorescence intensity of 2′,7′-dichlorodihydroflurescein (H₂DCFDA) (Invitrogen). Briefly, cells were treated with H₂DCFDA for 30 min. At least 2×10⁴ cells were analyzed by a FACSCalibur flow cytometer (Becton-Dickinson, San Josè, CA, USA), with laser excitation set at 495 nm and a 525-nm emission filter to detect green fluorescence. The negative control was obtained omitting fluorescence probe-mix from the reaction and auto-fluorescence was estimated. To study DNA content, cells were treated with propidium iodide (PI). At least 10⁵ cells were analyzed by FACSCalibur previously calibrated by CaliBRITE 3 beads (Becton-Dickinson), with laser excitation set at 488 nm and a 630-nm emission filter to detect red fluorescence. Level of 3′-terminal deoxy-transferase (TdT) by TUNEL method was used to detect apoptosis, following the manufacturer instructions (In Situ Cell Death Fluorescent kit, Roche Diagnostic S.p.A., Monza, Italy). Laser excitation was set at 488 nm and emission was at 550-nm for FITC. As an auto-fluorescence control, a sample treated with label solution but without TdT was carried out for each set of analyses. FCM histograms were analysed by the Windows Multiple Document Interface (WinMDI ver. 2.8, The Scripps Research Institute, La Jolla, CA, USA) dedicated software.

BSAO was purified from bovine blood as previously described (25). The purified enzyme moved as a single band on SDS/PAGE and all samples employed had a minimum specific benzylamine oxidase activity of 0.35 IU/mg, with IU defined as μmoles of substrate oxidized per min, assayed spectrophotometrically at 25°C by monitoring the formation of benzaldehyde at 250 nm absorbance (ɛ = 12,500 M⁻¹/cm⁻¹). The protein concentration was measured spectroscopically and from the 280 nm absorbance, assuming an absorption coefficient of 1.74 l g⁻¹/cm⁻¹.

Cell survival experiments were carried out using confluent cells that had been incubated for 24 h at 37°C with fresh culture medium. Cells were harvested with EDTA in phosphate buffer saline (PBS) and then by addition of trypsin solution in PBS, washed by centrifugation and resuspended in PBS supplemented with 1% bovine serum albumin (BSA) (Sigma). Freshly harvested LoVo WT and LoVo DX cells (10⁵/ml) were incubated at 37°C for varying time intervals in the presence of the following reagents, used alone or in combination: BSAO (1.03×10⁻⁴ μmoles/ml corresponding to 6.98×10⁻³ U/ml), spermine (0–6 μM), catalase (240 U/ml) from bovine liver (Sigma), ALDH (EC 1.2.1.5) from yeast (0.4 U/ml) and nicotine adenin dinucleotide (NAD⁺) (1.8 μg/ml; Boehringer-Mannheim, Mannheim, Germany). Spermine (Fluka, Buchs, Switzerland) was freshly prepared before each experiment and, if used, added last. Cells were then centrifuged, washed in PBS-BSA and finally resuspended in 1 ml PBS-BSA. The cells were then plated in tissue culture-coated Petri dishes (60×15 mm) and incubated at 37°C. Cytotoxicity was evaluated using a colony survival assay, thus determining the ability of cells to reproduce and form macroscopic colonies (>50 cells). After three weeks, colonies were fixed with 96% ethanol, stained with methylene blue and counted manually. Percentage cell survival was determined as the ratio between the mean number of colonies in treated and control samples.

Total RNA was extracted from the hippocampus and frontal cortex with TRizol reagent (Invitrogen) and subjected to DNaseI treatment (Promega) according to the manufacturer’s instructions. Two micrograms of total RNA were then used for cDNA synthesis, using SuperScript II (BRL Life Technologies) and random hexamer primers according to the manufacturer’s instructions. One microliter of cDNA was used for amplification using the following primers: mGlu2 receptor: Bcl-2 forward, 5′-CTA CAGTGATGTCTCCATCC-3′, reverse, 5′-AAAGCCTCAATG CCTGTCTC-3′; mGlu3 receptor: forward, 5′-CAAGTGAC TACAGAGTGCAG-3′, reverse, 5′-CTGTCACCAATGCTCAG CTC-3′; β-actin: BAX forward, 5′-TGAACCCTAAGGCCAA CCGTG-3′ reverse, 5′-GCTCATAGCTCTTCTCCAGGG-3′. Real-time quantitative PCR was performed using a 2X Supermix mixture (Bio-Rad) containing the double-stranded DNA Binding fluorescent probe SYBR Green and all necessary components except primers. Quantitative PCR conditions included an initial denaturation step of 94°C/10 min, followed by 40 cycles of 94°C/15 sec and 55°C/15 sec. Standards, samples and negative controls (no template) were analyzed in triplicate. Concentrations of mRNA were calculated from serially diluted standard curves simultaneously amplified with the unknown samples and corrected for β-actin mRNA levels.

Significant difference at p<0.05 was evaluated by the one-way ANOVA, followed by the multiple comparison Tukey post-hoc test (SPSS-11 statistical dedicated software - SPSS Inc., Chicago, IL, USA). Whisker box-plot graphs were obtained by open source software Gnumeric (Linux environment). All experiments were repeated three times unless otherwise indicated.

In previous studies, mSMOX activity was described to induce chronic sub-lethal DNA damage, with a 3-fold increase in oxo⁸dG residues, but no increase in cell mortality. Upon 2- and 4-Gy doses of X-irradiation, SMOX transfected cells were sensitized and more prone to die than mock transfected cells. Treatments with increasing doses of MDL abolished such radiosensitive predisposition (15,16). However, the level of X-ray delivered has to be considered as high doses, thus belonging to the LNT (linear no-threshold) theory of a linear dose-response. In the present study, we delivered a low dose of X-rays (10 cGy) (Fig. 1A, bar-graphs) with the relative cell cycle compartment composition, as determined by FCM. To confirm previous data, mSMOX alone does not apparently alter cell cycle and/or cell mortality, even in the presence of low dose irradiation at 6 and 24 h after exposure. Interestingly, when proliferation rates were detected by the more sensitive MTT assays at 6 h, mSMOX associated with a hypersensitive dose-effect curve at low dose and with an adaptive response at higher dose (Fig. 1B).

To dissect the influence of chronic sub-lethal DNA damage and DNA repair mechanisms, mSMOX was ectopically overexpressed in proficient Chinese hamster AA8 cell line and both deficient base-excision-repair EM9 cell line and deficient transcription-coupled nucleotide-excision-repair UV61 cell line to represent cellular models of priming damage dose of ROS. In Fig. 2A, proliferation MTT assays are represented as dose-effect curves related to low dose irradiation at 1 and 10 cGy, both determined at 6 and 24 h after exposure. In the AA8 and BER deficient EM9 cell lines, a hypersensitive reaction at 1-cGy exposure is detected at 6 h, independently from SMOX overexpression. At 24 h, the mock transfected AA8 cells followed a linear dose-effect curve. Contrarily, mSMOX transfected cells were resistant to both 1 and 10 cGy doses, showing a clear resistance to low dose radiation. In this case, mSMOX elicits an adaptive response rendering cells more reactive against DNA damage. In the EM9 cells, mSMOX does not alter the dose-effect curves of cellular response to irradiation at 24 h, being both more resistant than AA8 cells. However, the mSMOX overexpression provokes an additive damage with the BER deficiency upon low dose irradiation. In the UV61 cells, the 6 h hypersensitivity is registered only for mock transfected cells. At 24 h, cells keep a consistent proliferation rate independently of mSMOX and irradiation doses. The overexpression of mSMOX seems to deliver an earlier adaptive response at 6 h, although the proliferation-rates of UV61 cell line is much lower than AA8 and EM9 cell lines. According to the proliferation rates, we determined the course of apoptosis by means of TUNEL reaction. In Fig. 2B, representative FCM histograms are shown for the unirradiated and 24 h after 10-cGy exposure. In the AA8 cell line, the adaptive response delivered by mSMOX is clearly evidenced by the different reduced amounts of apoptosis at 24 h. In the EM9 cells, SMOX provokes more damage and, consistently, TUNEL reaction is more evident for mSMOX transfected cells. In the UV61 cells, TUNEL was barely detectable with no influence by mSMOX, probably also due to the low proliferation rates.

The interconversion metabolism of SPM by SMOX produces hydrogen-peroxide and substantial evidence has addressed this fundamental aspect of SMOX activity to cause intracellular ROS (14–16). In Fig. 3A the fold increase of ROS induced by the mSMOX overexpression is represented as box-plot graph for all cell lines (representative histograms are shown in Fig. 3B). The augmented levels of ROS were significantly higher in AA8 and EM9 cell lines when transfected with mSMOX. The ROS level in the UV61 cell line is not influenced by mSMOX. More likely, UV61 are very sensitive to ROS overproduction, being deficient in NER repair system and any subtle ROS variation is not compatible with cell life. Radiation provokes an almost three times ROS increase in all cell lines tested, reaching a hypothetical threshold, which may represent a sort of life-death barrier.

Fig. 4 shows the percentage of cell survival versus the time of exposure to purified BSAO (6.98×10⁻³ U/ml) in the presence of exogenous spermine (12 μM), with and without catalase, at 37°C. In the presence of BSAO and spermine alone higher cytotoxicity was observed in LoVo DX than in LoVo WT cells. The percent cell survival decreased in both cell lines with increasing exposure time, resulting in ~18% in LoVo WT (Fig. 4, curve a) and ~0.3% in LoVo DX cells (Fig. 4, curve b), after 60 min of incubation. In order to evaluate the contribution of each enzymatic oxidation product in the inhibition of cell growth, the experiments were performed in the presence of exogenous catalase, an enzyme which decomposes H₂O₂, or catalase and aldehyde dehydrogenase (ALDH) added simultaneously to the incubation mixture (data not shown). Catalase (240 U/ml) afforded a marked reduction of the cytotoxic effect, corresponding to ~80% cell survival, on LoVo WT and LoVo DX cells (Fig. 4, curves c and d, respectively), probably due to the clearance of hydrogen peroxide, formed in the catalytic reaction by the enzyme. The result showed that H₂O₂ was not the sole toxic factor and that other products of the enzymatic oxidative deamination were involved, such as aldehyde(s), including acrolein spontaneously formed from the aminoaldehydes (27), an aspect still debated. The addition of exogenous NAD-dependent ALDH (0.4 U/ml) metabolized the aldehyde form to the corresponding carboxilic acid and prevented the toxic effects of the aldehyde(s) or acrolein. In fact, after addition of both exogenous enzymes, catalase and NAD-dependent ALDH, cytotoxicity was completely inhibited throughout the 60 min of incubation (data not shown).

Cytotoxicity induced on human cancer cells by bovine serum amine oxidase (BSAO) and spermine is mainly attributed to H₂O₂ generated by the enzymatic reaction. Results obtained on colon adenocarcinoma LoVo WT and LoVo DX cells by real-time PCR experiments, showed an increase in mRNA levels for the BAX pro-apoptotic gene in MDR cells after treatment of both LoVo cell lines with 12 μM concentration of exogenous H₂O₂ (Fig. 5A). Whereas, LoVo cells treated with 12 μM concentration of exogenous acrolein, showed only a slight overexpression of BAX at the last stage of the reaction in MDR cells (Fig. 6A). In these experimental conditions, the pro-survival Bcl-2 gene did not reveal any variation after LoVo WT and MDR cell treatment with either exogenous H₂O₂ (Fig. 5B) or acrolein (Fig. 6B). Therefore, the enhancement in mRNA levels for the BAX pro-apoptotic gene due to H₂O₂ and acrolein was more marked in LoVo MDR cells than in LoVo WT ones, in agreement with the clonogenic assay obtained by the treatment of LoVo cells with BSAO in presence of 12 μM spermine concentration, as previously demonstrated by the authors.