Glioblastoma cell line (U373) was obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA) and grown, at 37°C and 5% CO2, in DMEM (Gibco, Grand Island, NY) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Euroclone, Ltd, UK), Gentamicin 20 ug/ml, 2 mM L-glutamine.
HIV-1 strain used in the preliminary experiment was R5-using Bal, and the HIV-1 strain used for the other experiments was X4-using IIIB. Both the viruses are provided by R. C. Gallo and M. Popovic at that time at the National Cancer Institute, National Institutes of Health, Bethesda, MD. The virus used for the experiments was ultra-centrifuged for two hours at 22,000 rpm at 4°C, stored in phosphate buffered saline (PBS) at -80°C.
Analysis of cellular apoptosis
U373 were seeded in Petri plates (60,000 cells/plate, Costar, Cambridge, MA) and, 24 hrs later, were exposed, where requested, to 1 mM NAC for 20 min. Then, 8,000 pg/ml of p24-gag HIV-1 IIIB were added to the medium (3 ml of medium culture). The cells were then incubated at 37°C in humidified air containing 5% CO2. On the day of analysis, the cells were gently detached with trypsin/EDTA (0.02%) and centrifuged at 1,600 rpm for 10 min. Pellets were washed with PBS, placed in ice, and permeated with ice-cold 70% ethanol for 30 min. The aliquots were centrifuged at 1,500 rpm for 10 min, the pellets were washed with PBS, incubated with propidium iodide (PI; 100 μg/ml, SIGMA-Aldrich, Germany) and RNase (250 μg/ml Qiagen, Mi, Italy) at 4°C for 2 h in the dark. Samples were then washed twice with PBS and PI-stained cells were analysed by monitoring the incorporation of intracellular PI with a FACScan flow cytometer. Results are from 3 separate experiments; 105 events were collected for each sample. Data were acquired and analysed by the Lysis II program (Becton Dickinson, Buccinasco, Mi, Italy).
Cells for electron microscopy (1 × 106/ml) were fixed in 2.5% glutaraldehyde in PBS, pH 7.4, at 4°C and then washed for 2 times in PBS and post-fixed in osmium tetroxide, 1.33% for 2 h at 4°C. After several washes in PBS, the cells were dehydrated in graded alcohol, transferred into toluene, and embedded in Epon 812 resin. The resin was allowed to polymerize in a dry oven at 60°C for 24 h. Thin sections were cut and stained with toluidine blue, and examined on an Axioscope microscope. Ultrathin sections were cut on a Reichert microtome using a diamond knife, stained with uranyl acetate/lead-citrate, and evaluated and photographed on a Philips electron microscope CM 10 (Philips Electronic Instruments, Mt. Vernon, NY).
Evaluation of telomeric length
cells metaphases, obtained treating samples for 2,5 hours with colchicine (10-6 M), were treated with hypotonic KCl buffer (0.075 M) and successively fixed with methanol: acid acetic (3:1). Cells were therefore seeded onto glass slides and stored at RT for 48 hours. Metaphase chromosomes were analyzed by Quantitative-Fluorescent In Situ Hybridization (Q-FISH) with peptide nucleic acid (PNA)-telomeric probe. Briefly, after washing with Tris Buffered Saline (TBS), slides were fixed in formaldehyde (3.7%), treated with proteinase K, dehydrated through a series of ethanol rinses (70%–85%–100%) and air-dried. Probe mixture containing Cy-3-conjugated (C3TA2)3 peptide nucleic acid (PNA) and Cy-3 centromeric-PNA(chromosome 2) probe (DAKO, Glostrup, Denmark) was added to the slides, and a DNA/probe co-denaturation (3 min at 80°C) was carried out. After hybridization for 2 hours at room temperature, slides were washed in 4 × SSC + 0.01%Tween20 for 5 min at 65°C and dehydrated in an ethanol series (70%–85%–100% for 2 min). Finally, slides were counterstained with DAPI in Vectashield (Vector Laboratories, Burlingame, CA).
Metaphases were captured and karyotyped using dedicated software. Telomeres length of each chromosome was measured by ISIS/Telomere software (MetaSystems) which allows precise measurement of single telomere signal. Centromere 2 was used as reference signal allowing to calculate the Telomere/Centromere ratio (T/C ratio). Each data was shown as percentage of T/C ratio. Statistical analysis was calculated by comparing >1800 telomere values measured at least 10 metaphases for each experimental point.
Estimation of telomerase activity
the PCR-based telomeric-repeat amplification protocol (TRAPeze ELISA Telomerase detection Kit) in accordance with the method proposed by Kim et al.,  was used to evaluate the activity of telomerase. This procedure is separated in three steps: 1) Extract Preparation, proteic extract of untreated and HIV-treated cells was obtained by using 1× CHAPS lysis buffer (10 mM Tris HCl, 1 mM MgCl2, 1 mM EGTA, 0,1 mM PMSF, 5 mM β-Mercaptoethanol, 0,5% CHAPS, 10% glicerol) 2) TRAP extension/amplification, where telomerase present in proteic extract, adds a number of telomeric repeats (GGTTAG) onto the 3' end of a biotinylated substrate oligonucleotide (b-TS); the extended products are then amplified by PCR. 3) Detection (ELISA). PCR products were analysed and the absorbance (A) was evaluated by the microplate reader. The difference between the absorbance for the sample and heat-treated sample was indicated as ΔA. When ΔA > 0.150 the sample is defined telomerase positive.
Measurement of GSH and GSSG
we measured the oxidative stress analysing the GSH value and the GSH/GSSG ratio, as is already well documented by the literature [49–51]. Specifically, intracellular cell GSH and GSSG content were determined by High-Performance Liquid Chromatography (HPLC) according to Reed et al. . Briefly, U373 cells were gently scraped off, washed and harvested by centrifugation at 2,000 rpm in a refrigerated centrifuge. Cell samples were suspended in phosphate-buffered saline and then lysed by repeated cycles of freezing and thawing under liquid nitrogen. Proteins were precipitated by adding sodium metaphosphoric acid (MPA) to a final concentration of 5% (w/v). A 0.5 ml aliquot of the clear supernatant, was treated immediately with 50 ul of a fresh aqueous solution (4 umol) iodoacetic acid and then neutralized with an excess of NaHCO3 (dry powder). After 60 min in the dark at room temperature, 0.5 ml of an alcoholic solution of 1-fluoro-2,4-dinitrobenzene (1.5 ml/98.5 ml absolute ethanol) was added and allowed to react overnight in the dark . Aliquots (100 ul) of the reaction mixtures were subjected to HPLC analysis. Protein levels of the cell samples were determined by the Bredford method [52, 53].
Trypan blue-exclusion test of cell viability
The dye-exclusion test was used to determine the number of viable cells after exposure of astrocytes to NAC. At days 1, 3 and 5, astrocytes were trypsinized, exposed to dye, and then examinated visually to determine whether cells take up or exclude dye. The live cells that possess intact cell membranes exclude trypan blue, whereas dead cells do not .
All the results were given as mean ± sem. Data are from 3 separate experiments, each experiment was run in triplicate. These results were performed using ANOVA followed by Student-Newman-Keuls unless specified (n = 3 different experiments). P < 0.05 was considered statistically significant.