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Human Molecular Genetics, 2003, Vol. 12, No. 18 2301-2309
DOI: 10.1093/hmg/ddg242
© 2003 Oxford University Press

DNA damage induced by polyglutamine-expanded proteins

Paola Giuliano1, Tiziana de Cristofaro2, Adelina Affaitati1, Grazia M. Pizzulo1, Antonio Feliciello3, Chiara Criscuolo4, Giuseppe De Michele4, Alessandro Filla4, Enrico V. Avvedimento3,* and Stelio Varrone3

1BioGem Consortium and 2IEOS-CNR c/o Department of Molecular and Cellular Biology and Pathology, 3Department of Molecular and Cellular Biology and Pathology and 4Department of Neurology, Federico II University, Naples, Italy

Received April 17, 2003; Accepted June 11, 2003


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
We have developed stable cell lines expressing green fluorescent protein fusion proteins containing polyglutamine repeats of various lengths under tetracycline control. The expression of the expanded (43Q) repeat protein resulted in aggregate formation in a time-dependent fashion. The accumulation of aggregates did not induce apoptosis, although the survival of these cells was critically dependent on the presence of serum and growth factors. However, the expression of 43Q expanded protein strongly activated the ataxia telangiectasia mutated kinase/ATM and Rad3-related kinase (ATM/ATR)-dependent DNA damage response, as shown by selective phosphorylation of ATM substrates. This activation was dependent on 43 CAG protein expression, reversible and sensitive to caffeine and reducing agents. Similarly, we found phosphorylated ATM substrates in fibroblasts from Huntington's disease or SCA-2 patients. Oxidative stress induced accumulation of ATM/ATR phosphorylated protein in HD and SCA-2 patients, but not in normal controls. Furthermore, a significant phosphorylation of H2AX was shown by fibroblasts from patients. We conclude that polyglutamine induces ATM/ATR-dependent DNA damage response through accumulation of reactive oxygen species. ATM activation can be used to monitor the disease in vivo.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Polyglutamine diseases are late-onset, neurodegenerative disorders arising from the expansion of an unstable CAG repeat in the coding DNA sequence, which is translated in a series of glutamine residues in the protein. A growing number of inherited neurodegenerative disorders, caused by polyQ expansion, includes spinobulbar muscular atrophy (SBMA) (1,2), Huntington's disease (HD) (3), dentatorubral pallidolusian atrophy (DRPLA) (4,5), and six spinocerebellar ataxias (SCA-1, 2, 3, 6, 7, 17) (6–14). Although the proteins harbouring the polyglutamine tracts are unrelated to each other and the neurons affected are different, it is likely that a common patho-genic mechanism might be responsible for the pathology of this group of diseases (15). Evidences have been provided that mitochondrial dysfunction plays a role in polyQ-induced cell death (16–18).

Recently two neurodegenerative diseases, spinocerebellar ataxia with axonal neuropathy (SCAN1) and the ataxia ocular apraxia (AOA1), have been linked to defects in DNA repair (19–21). The genes mutated are DNA repair genes. These disorders share several neurological features with ataxia telangiectasia (AT) syndrome, including early onset ataxia and cerebellar atrophy (22,23).

The aims of our study were: (1) to set up an inducible cellular model which summarizes the features of expanded polyQ-containing proteins; and (2) to analyze the biological effects of polyQ protein expression. To this end, we have constructed PC12 cell lines, conditionally expressing a synthetic protein containing the green fluorescence protein, GFP, fused to a stretch of 17 or 43 glutamine residues and to the HA (haemoagglutinin influenza human virus) epitope.

We report here that polyQ expanded protein induced a severe stress response of the cells. We also found that polyQ protein expression induced a robust and reversible activation of AT mutated kinase/ATM and Rad3-related kinase (ATM/ATR)-dependent DNA damage response. This response was also detected ex vivo in fibroblasts derived from patients with HD or SCA-2 diseases.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The expression of polyQ protein causes time-dependent formation of aggregates
GFP fusion proteins were generated by linking GFP and the HA epitope with 17 or 43 glutamine residues (24). These proteins were expressed under the control of TRE (tetracycline responsive element) promoter in a rat pheochromocytoma cell line, PC12 cells, carrying an engineered version of tetracycline-controlled transactivator (tTa). Single clones were characterized for induced-expression of the fusion proteins, following removal of the tetracycline analogue doxycycline (Dox). The best responders were further analysed. Very low basal levels of the fusions proteins were detected in the cells grown in the presence of doxycycline. Upon removal of doxycycline, the TRE promoter was activated and an intense GFP fluorescence was detected in cells transfected with the two expression vectors encoding the 17Q or 43Q fusion proteins. HA-17Q-GFP expressing cells showed a diffuse signal throughout the cytoplasm and nucleus and did not form aggregates (Fig. 1A). The cells expressing the expanded polyQ protein (HA-43Q-GFP) showed initially (1–2 days following doxycycline removal) a diffuse GFP fluorescence, followed by a progressive formation of aggregates. Three days after the induction, intranuclear and perinuclear aggregates were visible in 30–40% of the cells. After 6 days, they were present in 100% of the cells (Fig. 1A). Immunoblot analysis with an anti-HA antibody confirmed the fluorescence data (Fig. 1B). In the presence of doxycycline, a faint band HA-positive could be detected in HA-17Q-GFP and HA-43Q-GFP cells. The signal increased ~50-fold following the removal of doxycycline. Both HA-17Q-GFP and HA-43Q-GFP proteins of the expected molecular weight were present in the soluble fraction (Fig. 1B). We noticed that only in 43Q expressing cell extracts was there a high molecular weight HA-positive band in the stacking gel. This band appeared 3 days after removal of the drug and steadily increased after 6 days (Fig. 1B). These bands reacted with both the anti HA and anti GFP antibody, indicating that the proteins present in the aggregates were not cleaved (data not shown) (24). However, we cannot rule out that a mixture of cleaved and uncleaved fragments constitutes the nuclear aggregates.



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  		Figure 1. Time-dependent expression of polyglutamine-GFP fusion proteins in inducible PC12 cell lines. (A) Phase contrast (left) and fluorescence images (right) of PC12 cells stably transfected with either HA-17Q-GFP or HA-43Q-GFP and grown in the absence of Doxycycline for the indicated times. The localization of GFP-fusion proteins was examined by fluorescence microscopy. (B) Western blot of total cell extracts with anti-HA antibody, which recognizes the epitope-tagged fusion proteins. Fifty micrograms of extracts were fractionated on a 10% polyacrylamide gel, blotted and incubated with the antibody (1 : 5000). The arrows indicate the apparent molecular weight of fusion proteins. The high molecular weight bands visible in the stacking gel are only seen in extracts of HA-43Q-GFP cells induced for 6 days.

 
Expression of expanded polyQ proteins severely impairs cell response to stress
The data reported above indicate that the expression of the 43Q containing protein induces the formation of nuclear aggregates, which are critically dependent on the length and the time of expression of CAG repeats (25). Aggregates have been linked to neuronal cell death in vivo and in vitro (26–28). However, under normal growth conditions, we did not appreciate differences in the growth rate and DNA synthesis of 43Q expressing cells (data not shown). To find out if expanded polyQ proteins influenced the response of the cells to stress, we have investigated the sensitivity of these cells to growth factors and serum starvation. Figure 2A shows that PC12 cells in low serum undergo cell death in a time-dependent manner. No difference in cell death was noted among different cell lines in the presence of doxycycline. When the HA-43Q-GFP protein was induced, a greater and significant fraction of cells underwent apoptosis compared to un-induced cells or HA-17Q-GFP expressing cells. Twenty-five per cent of HA-43Q-GFP expressing cells underwent apoptosis after 48 h of serum starvation. Seven to eight per cent of control PC12 cells or lines expressing the HA-17Q-GFP protein underwent apoptosis. Under these conditions, treatment of the cells with a caspase inhibitor, Z-VAD-FMK, completely blocked cell death induced by serum withdrawal (data not shown).



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  		Figure 2. PolyQ-expressing cells are more susceptible to stress-induced apoptosis. (A) Time-course of apoptosis induced by serum-deprivation in cells transfected with HA-43CAG-GFP and HA-17CAG-GFP constructs. The fraction of cell death was determined as described in Materials and Methods. The values are represented as fold increase over the basal level in growing conditions. 500–1000 cells per samples were scored in three independent experiments (P<0.001: uninduced versus induced cells; P<0.001: 17 CAG versus 43 CAG). The doubling time was 48 h for HA-43CAG-GFP and HA-17CAG-GFP cells in the absence or presence of doxycyclin. (B) JNK activation in induced cells. JNK activation was determined by immunoblot with anti-P-JNK antibody. Uninduced and induced cells were serum-deprived for the indicated times. The blots were stripped and reprobed with the anti-JNK antibody. (C) Comparison of ROS levels in HA-43CAG-GFP transfected cells which were serum-deprived for the indicated times. The values are reported as fold increase over the basal level of uninduced cells in growing conditions. (D) Western blot analysis performed on cytosolic fractions from HA43Q-GFP cells incubated with anti-cytochrome c antibody. The same blot was probed with a-tubulin antibody to normalize the proteins loaded in each lane. In all the experiments fusion proteins were induced for 3 days (HA-43CAG-GFP) and 6 days (HA-17CAG-GFP) in absence of doxycycline before serum withdrawal.

 
To get an insight on the cell signaling pathways involved, we measured the activation of stress activated protein kinases. JNK (c-jun N-terminal kinase) activation has been implicated in neuronal cell death induced by environmental stress and by glutamate-mediated excitotoxicity (29,30). Furthermore, the activation of JNK has been reported in a neuronal cell line expressing full-length huntingtin with 48Q repeats (31). Figure 2B shows that serum deprivation induced a sustained activation of JNK only in 43Q expressing cells. 43Q un-induced cells and 17Q expressing cells (control cells), under the same conditions, did not activate JNK.

Since the mitochondria are the main effectors of stress-induced cell death (32), we have measured reactive oxygen species (ROS) levels and cytochrome C release in 43Q transfected cells. Higher ROS levels were found in growing HA-43Q-GFP expressing cells compared with parental cells or HA-17Q-GFP expressing cells. A sustained and further increase of ROS levels was found after 6 h of serum deprivation (Fig. 2C). Under the same conditions, 17Q cells or 43Q cells treated with doxycycline did not increase cellular ROS (Fig. 2C and data not shown). ROS accumulation was associated with cytochrome c release in the cytosol. Figure 2D shows that cytochrome c was released in the cytosol of cells expressing the 43Q protein as early as 12 h following serum withdrawal while in doxycycline treated cells the release of cytochrome C was late and scant. Inhibition of ROS elevation even at earlier times with NAC prevented cytochrome release later (data not shown).

Taken together these findings suggest an increased sensitivity to stress-induced cell death by expanded polyQ containing proteins and indicate a specific mitochondrial involvement.

Activation of DNA damage checkpoint by polyQ proteins
Oxidative stress is the principal source of DNA lesions. ATM (ataxia telangiectasia mutated kinase), ATR (ATM and Rad3-related kinase) and DNA-PK (DNA-dependent protein kinase) are serine/threonine protein kinases belonging to phosphatidylinositol (3) kinase family that are involved in DNA repair and recombination. These three related enzymes have similar target sequences and a general consensus motif has been defined (23,33–35). Using a phospho-Ser/Thr antibody generated against the peptide L-S/T-Q (specific for phosphorylated ATM/ATR substrates) we analysed the accumulation of ATM phosphorylated substrates. Figure 3A shows an immunoblot of nuclear proteins of PC12 cells. A 32 kDa band appeared very early (1 day) following induction of the expanded polyQ protein. After 4 days in the absence of doxycycline, a new band of 60 kDa appeared (Fig. 3A, 4 and 6 days). At this time point the addition of doxycycline for 1 day to repress polyQ protein expression reduced both bands (Fig. 3A, lane indicated by 6/1). These bands disappeared when the cells were treated with caffeine, an inhibitor of ATM and ATR kinases (Fig. 3C). The bands were differentially sensitive to caffeine. The 32 kDa band was highly sensitive since it disappeared after 2 h of treatment with 2 mM caffeine. On the other hand, the 60 kDa band appeared to be more resistant to caffeine treatment. Cells expressing the 17Q protein did not accumulate any specific band recognized by the antibody above indicated after 6 days of induction (Fig. 3A).



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  		Figure 3. The expression of HA-43Q-GFP fusion protein increase the ATM activity. (A) Time-course of HA-43Q-GFP induction upon doxycycline removal. A time point of HA-17Q-GFP expression is reported as control. The nuclear extracts were prepared as described in Materials and Methods and fractionated on a 12% polyacrylamide gel, blotted and incubated with phospho-Ser/Thr ATM/ATR substrates antibody. The lower part of the same gel was incubated with anti-H2A antibody to normalize the amount of loaded proteins. Three independent clones expressing HA-43Q-GFP and two clones expressing HA-17Q-GFP were analyzed. Both 60 and 32 kDa phosphorylated bands were present only in HA-43Q-GFP expressing clones. (B) Densitometric analysis of the lower band. Data are representative of independent experiments. (C) Caffeine sensitivity of HA-43Q-GFP transfected cells induced for 6 days in the absence of doxycycline. Western blot analysis was perfomed with anti-P-Ser/Thr ATM/ATR substrates antibody. Where indicated, the caffeine was added for 3 days and fresh solution was prepared every day. (D) P-ATM/ATR substrate sensitivity to treatment with a reducing agent, NAC. A stock solution of 250 mM of NAC was directly added to cells to give rise the final concentration. For each panel, one of three representative experiments is reported.

 
To further investigate if oxidative stress indeed contributed to polyQ-induced DNA damage, we tested the effect of a reducing agent, the N-acetyl-L-cysteine (NAC) on the accumulation of ATM/ATR substrates. We found (Fig. 3D) that the phosphorylation status of the two bands was greatly dependent on reactive oxygen species (ROS) levels, because 2 h of NAC treatment inhibited the accumulation of both bands.

The data shown above indicate a robust activation of the ATM/ATR-dependent DNA damage response following expanded polyQ proteins. Since expanded polyQ proteins are ubiquitously expressed, we asked whether fibroblasts from HD and SCA-2 patients showed ATM/ATR activation as PC12 cells expressing expanded polyQ proteins. We have extracted nuclear proteins from control, HD and SCA-2 fibroblasts stressed with 50 µm H2O2 for 15 min and immunoblotted them with the anti-ATM/ATR substrate antibody. Figure 4A shows that primary fibroblasts from healthy subjects stressed with H2O2 accumulated a 32 kDa band, which reacts with anti-ATM/ATR substrate antibody. The fibroblasts derived from HD and SCA-2 patients contain a band of the same size in basal conditions. Treatment with H2O2 significantly stimulated the 32 kDa band in cells derived from HD and SCA-2 patients. The 32 kDa band was also caffeine and NAC sensitive (data not shown), similarly to the 32 kDa band detected in PC12 cells (Fig. 3). Taken together these data strongly suggest that the 32 kDa band is the same protein found in PC12 cells, induced by expanded polyQ protein. We analysed two additional fibroblasts cell lines derived from one HD and one SCA-2 patient. The 32 kDa band was present in the nuclear extracts and was significantly stimulated by 50 µm H2O2 (data not shown).



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  		Figure 4. Activation of ATM in fibroblasts derived from HD and SCA-2 patients. (A) Nuclear extracts of fibroblasts from patients and from two different controls were analysed by western blotting with anti-P-Ser/Thr ATM/ATR substrates antibody. In the extracts from fibroblasts cells the antibody detect a band of 32 kDa which is stimulated by treatment with 50 µM H2O2. The normalization with anti-H2A antibody is shown. Densitometric analysis of the results is reported. Each histogram corresponds to a lane on the top of panel. Data are representative of three independent experiments performed on lines between V and IX passage. In each experiment the fibroblasts cells were harvested at the same passage number. The age of controls was comparable to that of the patients. (B) Immunofluorescence experiments performed with anti-{gamma}-H2AX antibody. Human primary fibroblats were seeded onto glass slides and treated with 50 µM H2O2. After 15 min the cells were fixed and incubated with primary antibody as described in Materials and Methods. Upper panel shows a 40x magnification. The lower panel presents a single cell magnification to show the distinctive features of H2AX foci. The percentage of cells of the patients showing brighter H2AX foci (compared to control cells) was ~60–70%.

 
A very early step in the cellular response to DNA double strand breaks is represented by phosphorylation of histone H2AX at the sites of DNA damage by ATM kinase (36). To obtain an independent evidence of ATM activation following DNA damage, fibroblasts from HD and SCA-2 patients were H2O2 treated and the presence of phosphorylated H2AX was monitored by immunofluorescence with an anti-{gamma}-H2AX antibody (Fig. 4B). A low and diffuse signal was detected in basal conditions in all fibroblasts with small appreciable differences between patients and controls (data not shown), but upon the stimulation with H2O2, many foci appeared in response to DNA damage induced by H2O2. In normal controls the signal was diffuse with small foci, while in the cells derived from the patients an intense fluorescence with larger foci was present. These data indicate that in fibroblasts derived from polyglutamine disease patients, there is a strong ATM/ATR-dependent DNA damage response compared with normal and healthy cells.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Polyglutamine neurodegenerative diseases are inherited disorders with an insidious onset in middle-age (mean ~40 years), with a strong inverse correlation between age of onset and length of CAG repeat. The clinical features of these diseases result from accumulation of cell damage, caused by mutated protein (37).

To better define the early events, we created an inducible system of PC12 cell line expressing HA-43Q-GFP fusion protein. By modulating the time of protein expression, we were able to identify and dissect the short and long term biological consequences following expanded polyQ expression.

DNA damage
Cells expressing expanded polyQ repeats accumulate intranuclear aggregates and suppress transcription of NGF- and cAMP-induced genes (38–40 and data not shown). These cells under standard culture conditions grow and proliferate as well as wild type cells. The accumulation of expanded polyQ proteins induces a significant stress to the cells (41). Mitochondria and the nucleus appear to be the primary sites. Recently, calcium defects have been found in lymphoblast mitochondria from HD patients. N-terminal mutant huntingtin has also been found in neuronal mitochondrial membranes (18). Under normal growth conditions, we find that polyQ expressing cells accumulate ROS. ROS can be efficiently scavenged in the mitochondria, cytoplasm and membrane by SOD enzymes (42). The only location where SOD enzymes are not present is the nucleus. This is an important site of stress in cells expressing expanded polyQ proteins, as shown by the formation of nuclear aggregates. The data reported above indicate a robust activation of ATM in these cells. Two different substrates are found phosphorylated in HA-43Q-GFP expressing cells. Both bands are caffeine sensitive (Fig. 3C) (ATM and ATR dependent) (43), although we cannot discriminate between these two kinases. The cells that accumulate these bands are not apoptotic cells, since we do not find any sign of cell death under these conditions, or gross impairment of the growth. The time course of phosphorylation of the bands shown in Figure 3 indicates that the 32 kDa substrate is rapidly induced and downregulated, while the 60 kDa band disappears slowly. The different turnover of phosphorylated ATM bands might be dependent on phosphatase activity present in the extracts. The sensitivity of these bands to N-acetylcysteine indicates that the primary cause of damage is the accumulation of ROS.

We have shown that ATM/ATR activation is a specific and reliable marker of polyQ protein expression in PC12 cells (Fig. 3). Similarly, fibroblasts derived from HD and SCA-2 patients show a robust activation of ATM/ATR as indicated by accumulation of a 32 kDa band, which reacts with anti ATM/ATR substrate antibody. We suggest that this band is the same protein found in PC12 cells expressing the polyQ expanded protein, because (1) it is dependent on polyQ protein expression; (2) it is caffeine- and NAC-sensitive. This band is stimulated by H2O2 also in cells derived from healthy subjects (Fig. 4). In several normal controls we have screened, the accumulation of the 32 kDa band was never comparable to that found in the cells derived from the HD or SCA-2 patients. It is worth noting that primary fibroblasts do not activate the 60 kDa band visible in PC12 cells. At present we cannot discriminate if the 60 kDa band is tissue specific, i.e. present only in neuronal cells, or it is less sensitive to oxidative stress.

Activation of ATM/ATR can be triggered by several stimuli that do not involve DNA damage (44). In vivo, at the sites of DNA lesions, accumulate nuclear foci, containing more than 20 proteins involved in DNA repair (45). The phosphorylation of H2AX and the presence of nuclear foci have always been found linked to DNA damage checkpoint dependent on ATM (46). To determine whether the cells derived from the HD and SCA-2 patients accumulate phosphorylated H2AX at DNA lesions (foci), we have stained normal and HD or SCA-2 fibroblasts with phospho-H2AX antibody and we have analysed the localization by fluorescence microscopy. The foci on the DNA of fibroblasts from HD or SCA-2 patients are significantly larger relative to normal healthy cells. Although H2O2 induces foci in healthy cells, the immunofluorescence signal in HD and SCA-2 fibroblasts is quantitatively and qualitatively different from that found in normal cells. The presence of phosphorylated H2AX and the accumulation of the 32 kDa band strongly indicate the presence of DNA damage in fibroblasts derived from the HD and SCA-2 patients.

The repair of DNA lesions is critical for the survival and genetic stability of mammalian cells. Several neurodegenerative disorders arise from mutations in DNA repair genes, but only the ataxia-telengiectasia disorder shows an increased genetic instability and a cancer predisposition (22,47). A deficiency of some components (for example tyrosyl phosphodiesterase, TDP1) of the DNA repair system in humans, does not predispose to neoplasia or dysfunctions in rapidly replicating tissues, but instead causes spinocerebellar ataxia with axonal neuropathy (21). We note, however, that polyQ-expressing cells efficiently repaired the DNA lesions, because they were not seriously impaired in short-term growth or survival. We cannot predict at the present the long-term effects of repaired DNA lesions on survival of post-mitotic cells.

Our results indicate that, in PC12 expressing expanded polyQ proteins or in fibroblasts derived from HD or SCA-2 patients, there is a significant activation of DNA damage response, which may represent a specific marker of polyQ diseases. We suggest that the accumulation of polyQ containing proteins imposes a considerable stress with an increased ROS accumulation (41). In long-term cultures or in vivo in post-mitotic neuronal cells, these events might seriously compromise transcription. Under these conditions, apoptosis could contribute to neurodegeneration. The extent and the rate of apoptosis may be different between neuronal and non-neuronal cells and among various types of neurons subjected to the same type of stress. For example, cerebellar granule or Purkinje cells are extremely sensitive to H2O2-induced apoptosis compared to cortical neurons (48). We suggest that ROS-induced DNA damage is amplified in specific neurons and might result in apoptosis later in the life of these cells. Moreover, a practical consequence of our finding is the possibility to monitor the progression of the disease by determining ATM/ATR activation in fibroblasts.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Plasmid construction
HA-17Q-GFP and HA-43Q-GFP cDNAs were excised from pCMV-HA-17Q-GFP and pCMV-HA-43Q-GFP constructs (24) using SacII/BamHI restriction enzymes. cDNAs were sub-cloned into pTRE-vector (Clontech) pre-digested with the same restriction enzymes. Nucleotide sequencing was performed as described (49) by using CEQ 2000 DNA Analysis System, Beckman automated sequencer, according to the manufacturer's protocol. cDNA vectors encoding GFP-fusion proteins were purified using QIAGEN Plasmid Maxi kit tips columns (Qiagen, Chatsworth, CA, USA).

Cell culture and transfection
Tet-Off PC12 cell line (Clontech, Palo Alto, CA, USA) were maintained in Dulbecco's modified Eagle's medium (DMEM, Gibco BRL) supplemented with 10% heat-inactivated horse serum (HS, Gibco BRL), 5% fetal bovine serum (FBS, Gibco BRL), 1 mM glutamine (Gibco BRL), 100 µg/ml streptomycin (Gibco BRL), 100 U/ml penicillin (Gibco BRL) and 100 µg/ml G418 (Gibco BRL) in a humidified 5% CO2 atmosphere at 37°C. PC12 cells were transfected at 40–60% confluence with 10 µg DNA using liposome-mediated transfection according to the manufacturer's instructions (Lipofectin Reagent, Gibco BRL). Positive clones were selected in complete medium containing: G-418, 100 µg/ml, hygromycin, 200 µg/ml (Gibco BRL) and doxycycline hydrochloride 10 ng/ml (Gibco BRL).

Cell death analysis
Cell death was analysed by staining with propidium iodide (PI). Briefly, cells were harvested at indicated times after treatment, washed twice with PBS 1x and incubated for 10 min with propidium iodide (50 ng/ml in PBS 1x, Sigma). Cells were washed and analysed by fluorescence microscopy using an Axiover microscope IX70 (Olympus). The data are presented as a percentage of propidium iodide positive cells on total number of cells (700 cells were considered in each of three independent experiments).

Western blot analysis
Cells were homogenized in lysis buffer (40 mM Hepes pH 7.5, 120 mM NaCl, 5 mM MgCl2, 1 mM EGTA, 0.5 mM EDTA, 0.6% Triton X-100) containing protease (Complete Tablets, EDTA-free, Roche) and phosphatase (20 mM ß-glycerol-phosphate, 2.5 mM Na-pyrophosphate) inhibitors. Total lysate was cleared by centrifugation at 15 000g for 20 min. Protein concentration of supernatant was measured using Bio-Rad protein assay. The remaining insoluble pellet was dissolved in lysis buffer with 100 U Dnase for 15 min. For ATM/ATR substrates analysis, nuclear extracts were loaded according to supernatant concentration. Extracts were denatured and resolved by SDS–PAGE (12%) gel electrophoresis, transferred to a Hybond ECL nitrocellulose membrane (Amersham) by electroblotting at 250 mA for 2–3 h. For cytochrome C, cytosolic fractions were prepared as described (50).

The following primary antibodies were used: anti-HA (Roche), anti-SAPK/JNK and anti-P (Thr183/Tyr185)-JNK (Cell Signaling Technology), anti-cytochrome c (BD PharMingen), anti-{alpha}-tubulin (Sigma), anti-P(Ser139)-H2AX (Upstate Biotechnology), anti-P(Ser/Thr) ATM/ATR substrate (Cell Signaling Technology), anti-H2A (Cell Signaling Technology).

Anti-mouse IgG or anti-rabbit IgG coupled to peroxidase were used as secondary antibodies (Sigma). Chemioluminescent (ECL) signals were quantified by scanning densitometry using ‘Scion Image’ software.

Measurement of reactive oxygen species
The generation of reactive oxygen intermediates in cells was detected using dichlorodihydrofluorescein diacetate (H2DCFDA). After each treatment, cell pellets were washed three times with PBS and then with PBS containing H2DCFDA (5 µg/µl) (Molecular Probes). Fluorescence was determined by FACS analysis (fluorescence-activated cell sorter) with an excitation wavelength of 485 nm and an emission wavelength of 530 nm. The actual ROS levels were determined by subtracting GFP fluorescence in samples not treated with H2DCFDA. Under these conditions fluorescence was linearly dependent on the presence of H2DCFDA. Treatment of the cells with N-acetylcysteine (1 mM) completely suppressed H2DCFDA fluorescence. The values reported are the mean of at least three experiments.

Immunofluorescence
Primary fibroblasts grown on glass slides were fixed in 50% methanol/50% acetone for 2 h at -20°C. Following fixation, slides were washed three times for 5 min each in PBS and blocked in 10% FBS-PBS for 1 h at room temperature. Primary antibody ({gamma}-H2AX) was diluted at 1 : 500 in 1% bovine serum albumin-PBS and was incubated overnight at +4°C. After washing, secondary antibody (fluorescein isothiocyanate anti-rabbit) was incubated at 1 : 200 in 10% FBS-PBS for 1 h at room temperature. All images were captured with Axiocam microscopy (Zeiss) in the same conditions of brightness and contrast.


   ACKNOWLEDGEMENTS
 
This work was supported by a grant from MURST (Italian Department of University and Research), by a grant from BioGeM Consortium and by AIRC. We thank M. de Cristofaro and D. Rotoli for technical contribution and S. Salzano for the assistance in FACS analysis. We are also in debt to Dr M. R. Santillo for valuable help in ROS measurement and Dr V. Costanzo and Dr D. Grieco for stimulating discussions and advice on DNA damage checkpoints.


   FOOTNOTES
 
* To whom correspondence should be addressed at: Dipartimento di Biologia e Patologia Molecolare e Cellulare, Università ‘Federico II’, via Pansini 5, 80131, Napoli, Italy. Tel: +39 817463251; Fax: +39 817463252; Email: avvedim{at}unina.it Back


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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