5-Lipoxygenase regulates senescence-like growth arrest by promoting ROS-dependent p53 activation

Keywords: 5-lipoxygenase, oncogenic ras, p53, reactive oxygen species, senescence

5LO expression and activity in oncogenic ras-mediated stasis

We first examined the expression and activity of 5LO during stasis induced by oncogenic ras (Ha-RasV12) or sublethal doses of H₂O₂. As previously described, human (WI38, IMR-90 and LF1) and MEFs expressing Ha-RasV12 or treated with H₂O₂ (250 μM for 2 h) reduced [³H]thymidine uptake at subconfluent density (up to 85%) and were highly positive (up to 75%) for senescence-associated SA-β-gal by 10–15 days post-treatments (data not shown; Bischof et al, 2002; Gorbunova et al, 2002). Unlike H₂O₂, Ha-RasV12-arrested WI38 cells displayed a strong induction in 5LO, and in the senescence-related markers p53 and p21 (Figure 1A). IMR-90, LF1 and MEFs that underwent ‘static' by oncogenic ras also accumulated 5LO (Figure 1B). These changes were accompanied by a progressive increase in 5LO nuclear localization (data not shown). Accordingly, products of the 5LO pathway, 5-HETE and LTB4, were increased in conditioned media of Ha-RasV12-expressing fibroblasts by four- and three-fold, respectively. The production of the ‘total LTA4-derived metabolites' (the sum of LTB4, 20-OH-LTB4, Δ6-trans-LTB4 and 12-epi-Δ6-trans-LTB4) was also increased by 3.5-fold (Figure 1C). However, Ha-RasV12-arrested cells did not show appreciable changes in 12-HETE, a 12-LO metabolite, or cyclooxygenase (COX) metabolites, compared with control cells (data not shown). Ha-RasV12 upregulated two gene products of the 5LO cascade, cytosolic phospholipase A2 (cPLA2) and 5LO. In addition, oncogenic ras gave a 3.5-fold increase in COX2 mRNA, whereas it did not induce a significant change in LTA4 hydrolase, LTC4 synthase and 5LO activating protein (FLAP) mRNA levels (Supplementary Figure 1). H₂O₂ had no appreciable effect on 5LO pathway constituents. It therefore appears that the 5LO cascade is part of the signaling pathway of oncogenic ras-induced stasis.

5LO is critical for the ROS production and stasis induced by oncogenic ras

ROS production mediates Ha-RasV12-induced stasis (Lee et al, 1999). To determine whether 5LO pathway is involved in ROS generation and stasis induced by oncogenic ras, Ha-RasV12-arrested WI38 cells were preincubated with the 5LO inhibitors AA861, eicosatetraynoic acid (ETYA) and MK886, and the cPLA2 inhibitor arachidonyl trifluoromethyl ketone (ATK). For comparison purpose, we also used the nonspecific COX inhibitor indomethacin as well as N-acetylcysteine (NAC), a ROS scavenger, and diphenyleneiodonium chloride (DPI), an inhibitor of NADPH oxidase-like flavoenzymes, which are known to inhibit stasis (Lee et al, 1999; Haendeler et al, 2004). AA861 (1 μM) and MK886 (1 μM) potently suppressed (up to 70%) 5-HETE formation in Ha-RasV12-stimulated WI38 cells. ATK (10 μM) also reduced by 60% the amount of 5-HETE, and high concentrations of ETYA (35 μM) were required for similar inhibition (Supplementary Figure 2A). The concentration of these inhibitors corresponded to approximately twice the previously determined ID₅₀. As expected, Ha-RasV12-induced 5LO activity was not significantly affected by indomethacin (10 μM), NAC (2 mM) and DPI (15 μM) (Supplementary Figure 2A). We observed similar findings when LTB4 determination was used instead of 5-HETE (data not shown). Under these conditions, the cPLA2 and 5LO inhibitors markedly decreased Ha-RasV12-induced ROS and SA-β-gal accumulation. By contrast, indomethacin had little or no effect (Supplementary Figure 2B). As expected, NAC and DPI diminished ROS and SA-β-gal accumulation elicited by oncogenic ras (Supplementary Figure 2B), whereas AA861, ETYA, MK886 and ATK did not influence ROS and SA-β-gal accumulation after H₂O₂ treatment (data not shown). This suggests that cPLA2-catalyzed synthesis of arachidonic acid and subsequent metabolism of arachidonic acid by 5LO are involved in the Ha-RasV12 signaling to ROS generation and stasis.

To test this idea, Ha-RasV12-induced stasis was analyzed in 5LO-deficient (5LO^−/−) MEFs. Ha-RasV12-infected wild-type MEFs ceased to proliferate at subconfluent density, whereas Ha-RasV12 expression had a minor and delayed effect on proliferation of 5LO^−/− MEFs (Figure 2A). The frequency of SA-β-gal-positive cells (data not shown) and DCF fluorescence (Figure 2B, left panel) were significantly lower in oncogenic ras-expressing 5LO^−/− MEFs than in wild-type MEFs. Similar results were obtained when the formation of superoxide anion (O₂⁻) was determined by measuring the chemiluminescence of the metabolized lucigenin. In fact, Ha-RasV12 expression in wild-type MEFs resulted in an 8.5-fold increase in O₂⁻ formation compared to control cells, whereas it caused only a two-fold increase in 5LO^−/− MEFs (Figure 2B, right panel). The addition of pro-oxidant tert-butyl-H₂O₂ (t-BTH) rescued the antistatic effect in Ha-RasV12-infected 5LO^−/− MEFs (Figure 2C). Thus, exogenous ROS can cooperate with Ha-RasV12 to overcome the antistatic effect of 5LO deficiency. Extension of lifespan after oncogenic ras transduction into 5LO^−/− MEFs was followed at later passages (at day 12 until day 16) by apoptosis of a large fraction of Ha-RasV12-overexpressing 5LO^−/− MEFs (Figure 2D). Therefore, oncogenic ras may promote either stasis or apoptosis depending on the expression levels of 5LO in the cell.

5LO pathway induces telomerase-independent stasis by increasing intracellular ROS

To further evaluate the role of 5LO pathway in stasis, we introduced a green fluorescent protein (GFP)-tagged 5LO or catalytically inactive 5LO mutant (H367Q) into WI38 fibroblasts using a pBabe-based vector. When WI38 fibroblasts were not selected with puromycin following 5LO infection, 5LO did not induce significant SA-β-gal accumulation and growth arrest. In contrast, after selection, 5LO caused stasis within 10-15 days from infection (Figure 3A and B). ROS levels induced by 5LO, without selection, increased around two-fold over controls, whereas WI38 fibroblasts selected following 5LO infection had a greater than five-fold increase in ROS levels (Figure 3C). These different cell responses were linked with the increased 5LO protein and LTB4 formation observed (Figure 3D). We observed similar results when 5-HETE determination was used instead of LTB4 (data not shown). Notably, overexpression of H367Q was completely ineffective. Thus, quantitative differences in 5LO expression and activity correlated with ROS induction and stasis. Neither cPLA2 nor COX2 overexpression provoked SA-β-gal activity and reduction of [³H]thymidine incorporation, although these proteins were efficiently expressed (data not shown).

To determine what ROS are actually formed in 5LO-expressing cells, we assessed the levels of O₂⁻, H₂O₂, NO₂⁻ and lipid peroxidation. Like oncogenic ras, 5LO expression resulted in a nine-fold increase in both O₂⁻ and H₂O₂ formation compared to H367Q or control cells. Moreover, 5LO as well as Ha-RasV12 caused a four- and three-fold elevation in the level of NO₂⁻ and lipid peroxides, respectively (Figure 4A). Interestingly, COX2 overexpression also increased lipid peroxidation and NO₂⁻ formation; however, no generation of oxygen radicals (O₂⁻ and H₂O₂) has been detected (data not shown).

To better understand the importance of the rise in ROS levels (O₂⁻ and H₂O₂, mainly) in the development of the 5LO-induced stasis, we cultured 5LO-expressing cells in varying oxygen environments, an established method to regulate ROS production (Parrinello et al, 2003). 5LO-expressing cells maintained in the standard tissue culture environment of 20% oxygen undergo near complete growth arrest. By contrast, lowering ambient oxygen to 3% enabled cells expressing 5LO to continue to proliferate (Figure 4B). In addition, 5LO-mediated growth arrest and SA-β-gal accumulation were rescued by the antioxidant NAC or by AA861 in 20% oxygen (data not shown and Figure 4C). Therefore, 5LO pathway generates ROS, O₂⁻ and H₂O₂ in particular, which in turn promotes stasis.

To test whether 5LO-induced stasis is bypassed by constitutive expression of catalytically active human telomerase (hTERT), we coinfected hTERT-expressing human fibroblasts along with the respective 5LO constructs. In the case of 5LO, no differences in the inhibition of cell proliferation (Supplementary Figure 3) or SA-β-gal accumulation (data not shown) were observed. Thus, the induction of stasis by 5LO-linked cascade is hTERT independent.

5LO elicits stasis via p53-dependent mechanism

The p53/p21 pathway contributes to stasis induced by oncogenic ras (Pearson et al, 2000). 5LO-expressing WI38 cells showed an accumulation of p53 (four- to five-fold) and p21 (five- to six-fold), a decrease of pRb phosphorylation and they did not show any difference of PML and p16 protein levels in comparison to controls (Figure 5A). To determine the role of p53 and p21 in 5LO-induced arrest, we infected MEFs derived from p53- (p53^−/−) or p21-deficient (p21^−/−) mice with pBabe-5LO vectors. 5LO overexpression reduced cell proliferation in MEFs, but it did not induce growth arrest in both p53^−/− and p21^−/− MEFs (Figure 5B). Therefore, p53 and p21 are essential for 5LO-induced growth arrest. Expression of p21 is regulated by p53-dependent or -independent mechanisms as well as protein degradation (Fotedar et al, 2004). To investigate the mechanism by which 5LO increases p21 expression, 5LO-expressing wild-type and p53^−/− MEFs were then analyzed by immunoblotting. 5LO upregulated p21 in wild-type MEFs but not in p53^−/− MEFs (Figure 5C), indicating that p53 is crucial for 5LO-mediated p21 upregulation.

ROS production by 5LO-linked cascade regulates p53 phosphorylation and transactivation

To clarify the mechanism by which 5LO activated p53-dependent pathway, we first examined the expression of p53 and p21 proteins in Ha-RasV12-expressing wild-type and 5LO^−/− MEFs. Ha-RasV12 induced comparable amounts of p53 as well as p16 proteins in both cell types (Figure 6A), and these proteins were still induced in the presence of NAC or AA861 (data not shown). However, a slight, but reproducible, reduction of p21 upregulation occurred in 5LO^−/− MEFs (8- and 4.5-fold increase in wild-type and 5LO^−/− MEFs, respectively) (Figure 6A) or in wild-type MEFs treated with NAC or AA861 (data not shown). Thus, 5LO seems to regulate p53 activity rather than stability. To confirm this, we transfected Ha-RasV12-infected wild-type and 5LO^−/− MEFs with a luciferase reporter vector driven by the p21 promoter (p21-luc). In 5LO^−/− MEFs, Ha-RasV12-induced p53 transactivation was reduced compared to wild-type MEFs and the addition of t-BTH (10 μM) recovered Ha-RasV12-induced p53 transcriptional activity (Figure 6B). In wild-type MEFs, the enhancement of luciferase activity by Ha-RasV12 was significantly lower after treatment with either AA861 or NAC (Figure 6B). Thus, inhibition of 5LO cascade as well as addition of antioxidant NAC reduced oncogenic ras-induced p53 transcriptional activity.

p53 is activated by a variety of signals, some of which produce specific p53 post-translational modifications (Appella and Anderson, 2001). In particular, p53 phosphorylation accumulates during cellular senescence (Ferbeyre et al, 2000; Pearson et al, 2000). We compared the status of p53 phosphorylation during Ha-RasV12-induced stasis in wild-type and 5LO^−/− MEFs, as well as in 5LO-overexpressing cells. The phosphorylation of immunoprecipitated p53 was assessed using antibodies specific for phosphorylated Ser9, 15, 46 and 392. Ha-RasV12 increased p53 phosphorylation at Ser46 in wild-type or 5LO^−/− MEFs (Figure 6C). Ser46 phosphorylation was also increased in 5LO-expressing MEFs (Figure 6C). By contrast, Ser15 phosphorylation was observed in either Ha-RasV12- or 5LO-expressing cells, and it was dramatically reduced in 5LO^−/− MEFs (Figure 6C). Moreover, we could not detect any change in phosphorylation at Ser9 and 392 induced by Ha-RasV12 or 5LO expression (data not shown).

Further, we investigated whether Ser15 phosphorylation is relevant for p53 activation induced by 5LO. Transient transfection of wild-type p53 in p53^−/− MEFs activated the p21-luc in a dose-dependent manner (Figure 6D). p53 mutant defective for phosphorylation at Ser15 (p53S15A) yielded similar levels of promoter activation. Coexpression of 5LO or Ha-RasV12 increased the transcriptional activity of wild-type p53 but not that of p53S15A (Figure 6D), indicating that Ser15 phosphorylation is required for p53 activation induced by 5LO and Ha-RasV12. To examine whether Ser15 phosphorylation is dependent on 5LO-mediated ROS induction, we treated Ha-RasV12- or 5LO-expressing MEFs with NAC or AA861. Both treatments inhibited Ser15 phosphorylation (Figure 6E, left panel). Moreover, this phosphorylation was recovered in 5LO^−/− MEFs expressing Ha-RasV12 and treated with t-BTH (Figure 6E, right panel). Thus, 5LO-induced ROS production is important for a full p53 phosphorylation and activity during stasis.

5LO regulates the in vitro lifespan of human and murine fibroblasts

Human lung fibroblasts undergo oxidative crises during their culture history so that their growth arrest often represents a combination of replicative senescence and stasis (Atamna et al, 2000; Forsyth et al, 2003). To test the involvement of 5LO in mediating the oxidative crisis in human fibroblasts, WI38 cells were serially passaged until they entered a senescence-like growth arrest, and protein and conditioned medium samples were collected at several stages. Senescent cells had higher 5LO protein levels than young fibroblasts (Figure 7A). This increase was readily associated with accumulation of p53 and p21, p53 phosphorylation at Ser15 and pRb hypophosphorylation (Figure 7A). In addition, a significant increase in 5LO product formation was observed in conditioned media of late-passage and senescent cells (Figure 7B). 5LO upregulation is also shown in IMR-90 and LF1 senescence fibroblasts, whereas it did not occur in confluent WI38 cells (data not shown). ROS production increased during culture history, and incubation with AA861 reduced this increase in a dose-dependent manner (Figure 7C). Accordingly, AA861 and NAC strongly reduced Ser15 phosphorylation of p53 induced during cell culture history (Figure 7D). AA861 also extended the in vitro lifespan of WI38 cells in a dose-dependent manner (Figure 7E).

On the other hand, replicative senescence of MEFs is a primary consequence of severe oxidative stress, which causes extensive DNA damage (Parrinello et al, 2003). Increased 5LO expression and activity are also shown in senescence MEFs (data not shown) and 5LO^−/− MEFs showed a significant increase of in vitro lifespan compared to wild-type MEFs (Figure 7F). These data support the idea that 5LO may be a key component in mediating oxidative crisis in human and murine fibroblasts that, in turn, can promote p53 phosphorylation and stasis.

Cells and culture conditions

Normal human fibroblasts IMR-90 (ATCC CCL-186), LF1 (ATCC CCL-153) and WI38 (ATCC CCL-75) were cultured in Ham's F-10 medium supplemented with 15% fetal bovine serum (FBS), nonessential amino acids and sodium pyruvate (all from HyClone, Rome, Italy). 5LO^−/− MEFs were prepared from 5LO^−/− mice, whereas p21^−/− MEFs were prepared from embryos derived from crosses between heterozygous mice (Jackson, Bar Harbor, ME). MEFs were cultured in Dulbecco's modified Eagle's medium supplemented with 10% FBS and penicillin–streptomycin (50 U/ml). For H₂O₂ treatment (Gorbunova et al, 2002), we added to the culture medium 250 μM H₂O₂ for 2 h. Then, cells were washed once with phosphate-buffered saline (PBS) and a fresh culture medium was added. For infected cells (Ferbeyre et al, 2000), we selected with 1 μg/ml puromycin for 4 days. The day when drug selection was completed (4 days after infection or H₂O₂ treatment) was defined as day 0.

Chemicals and vectors

MK886, AA861, indometacin, ATK and ETYA were purchased from Cayman Chemical (Milan, Italy). DCF, DPI and NAC were from Sigma (Milan, Italy). All other chemicals were from standard sources and were molecular biology grade or higher. The retroviral Babe-puro-Ha-RasV12 and pBabe empty (B0) vectors were kindly provided by Dr GJ Clark (National Cancer Institute, MD). The plasmids coding wild-type p53 and p53S15A were described previously (D'Orazi et al, 2002), and were provided by Dr S Soddu (Regina Elena Cancer Institute, Rome, Italy). The cDNA of wild-type 5LO-GFP fusion protein or catalytically inactive 5LO mutant, H367Q-GFP fusion protein was previously reported (Romano et al, 2001; Catalano et al, 2004), and was subcloned into Babe-puro. DNA sequencing was performed to verify the correct insertion with a MegaBACE DNA analysis system by using a DYEnamic ET Dye Terminator Cycle sequencing kit (Amersham Corp., Milan, Italy). Details of the construct are available upon request. Retroviral stocks were generated by transient transfection of packaging cell line (PT67, Clontech, Palo Alto, CA) and stored at −80°C until use. Infection of cells with retroviral constructs, and culturing of transduced cells were performed by standard procedures (Serrano et al, 1997; Bischof et al, 2002).

Reporter assays

Infected WI38 fibroblasts (5–8 × 10³/cm² on 35 mm dishes) were transfected with 500 ng p21-Luciferase (p21-Luc) reporter plasmid (Catalano et al, 2004) and 0.1 μg pCMV-β-gal reporters using LipofectAMINE 2000 (Invitrogen, Milan, Italy). β-Galactosidase and luciferase activities were measured using Galacto-Light Plus™ (Tropix Inc., Bedford, MA) and Luciferase Assay Systems (Promega, Milan, Italy) according to the suppliers' instructions and as reported earlier (Catalano et al, 2004). Luciferase activity was then normalized with β-galactosidase activity.

Senescence analysis

Senescence was assessed using several assays. The growth curves were performed as described (Bischof et al, 2002). Cells were plated in triplicate at 2 × 10⁴ per well in 12-well plates. Relative cell numbers were estimated at various time points using a crystal violet incorporation assay, and the number of PDs (n) was calculated using the following equation:

where F is the number of cells at the end of one passage and I is the number of cells that were seeded at the beginning of one passage. Thymidine incorporation assay and SA-β-gal staining were performed as described (Gorbunova et al, 2002). Cells were subcultured on coverslips. When 70–80% confluent at 2 × 10⁴/cm², [³H]thymidine (10 μCi/ml) was added to the cells for an additional 36 h, fixed and subjected to autoradiography, and the percentage of cells incorporating [³H]thymidine was determined. These cells were also costained for SA-β-Gal. Cells were counted under the microscope, and a minimum of 500 cells were counted for each coverslip. The percent of SA-β-gal-positive cells from the total number of cells was calculated.

Real-time RT–PCR

Cells were collected after indicated times and washed with PBS. Total RNA was extracted with the RNeasy minikit from Qiagen (Milan, Italy). Total RNA (50 ng/μl) from different treatments was reverse transcribed with the TaqMan reverse transcription kit (PE Applied Biosystem, Milan, Italy). The cDNA was then subjected to real-time reverse transcription (RT)–PCR with SYBR Green PCR core reagents (Finnzymes, Keilaranta, Finland). The PCR were carried out in a Chromo4 sequence detector (Celbio, Milan, Italy). The primer sequences of all 5LO pathway constituents were determined by Laboratory Tools software analysis of Stratagene (Milan, Italy). Details of sequences and thermal cycle conditions are available upon request. The amplicons of genes were between 250 and 300 bp. Data were acquired and analyzed with the sequence detector Chromo4 software.

Protein expression

Immunoprecipitation and Western blot analysis were performed from whole cell lysates as previously described (Catalano et al, 2004). Antibodies against 5LO (Cayman Chemical, Milan, Italy), p53 (DO1, Santa Cruz, Milan, Italy), Ser15 (16G8), Ser46 p53 (Cell Signaling Tec., Milan, Italy), p21 (Ab1), RasV12 (OP38, Calbiochem, Milan, Italy), p16 (DCS-50, Novocastra), pRb, cPLA, COX2 (all by Cell Signaling Tec., Milan, Italy) and PML (PGM3, Santa Cruz) were used as probes and detected using enhanced chemiluminescence (Amersham Corp., Milan, Italy). To determine expression of the catalytically inactive H367Q mutant, Western blot analysis was carried out using anti-GFP monoclonal antibody (Calbiochem, Milan, Italy). As loading control, the blots were reprobed with an anti-β-actin antibody.

Measurements of 5LO products

Mono-HETEs released into cell conditioned media were determined using solid phase extraction and reversed-phase (RP) high-performance liquid chromatography (HPLC) procedures as described previously (Catalano et al, 2004). To determine LT isomers and metabolites, we also utilized RP-HPLC procedures described by Hosni et al (1991), with detection at 270 nm. Prostaglandins E1 (PGE1) and B2 (PGB2), 5-HETE, 15S-hydroxyeicosatrienoic acid (15-HETrE), LTB4 and other arachidonic acid metabolites used as standards for HPLC were from Cayman (Milan, Italy). Methanol and acetonitrile were of HPLC grade. Other reagents were of analytical grade or better. 5LO product formation is expressed as nanogram of 5LO products per milligram of protein. 20-COOH-LTB4 is not detected by RP-HPLC.

Measurement of ROS, superoxide, hydrogen peroxide, nitrite anion and 8-isoprostane generation

For measurement of ROS production, cells were incubated with 5 μg/ml of DCF for 30 min at 37°C, washed with PBS, trypsinized and collected in 1 ml of PBS, followed by fluorescence-activated cell sorter (FACS; Beckton Dickson FACScan) using Cell Quest 3.2 software (Beckton Dickson, Milan, Italy) for analysis. Values of mean green fluorescence intensity were used to plot graphs. Superoxide anion (O₂⁻) generation was determined by lucigenin (25 μM, final concentration) as previously described (Altmann et al, 2004). The chemiluminescence was recorded using a Micro Luminat Plus LB 96V (Berthold, Bad Wildbad, Germany) at intervals of 6 s (two cycles) in a total detection time of 3 min. The detected chemiluminescence was summarized over two intervals and plotted versus blank values. H₂O₂ concentration was determined as previously described (Moreno, 2003). Briefly, 2,6-dichlorophenolindophenol (DCPIP, 40 μM) was reduced by ascorbic acid, which attenuated the blue color. H₂O₂ in samples in the presence of a few microliters of horseradish peroxidase increased the absorbance (610 nm) owing to the reoxidation of DCPIP. Control reactions were performed by adding catalase. The 8-isoprostane was measured using the corresponding ELISA kit from Cayman Chemicals (Milan, Italy), and nitrite (NO₂⁻) accumulation in the cell-free supernatant, as an indicator of NO production, was measured using the Griess reagent as described elsewhere (Martinez et al, 2000). The production of NO₂⁻ was measured from a sodium nitrite standard curve freshly prepared in culture medium and expressed in nmol. The detection limit was 1 μM.

Annexin and propidium iodide fluorescent staining

Annexin and PI fluorescent staining was carried out as described previously (Rippo et al, 2004). Cells positively stained with annexin and not PI were considered apoptotic, and cells negative for the two dyes were considered live cells.

Statistical analysis

All values were expressed as mean±s.e.m. Comparison of results between different groups was performed by one-way analysis of variance and paired t-test using StatView 5.0 (NET Engineering, Pavia, Italy). A P-value ⩽0.05 was considered to be statistically significant.

Supplementary Figure 1

Supplementary Figure 2

Supplementary Figure 3

1. Altmann A, Poeckel D, Fischer L, Schubert-Zsilavecz M, Steinhilber D, Werz O (2004) Coupling of boswellic acid-induced Ca²⁺ mobilisation and MAPK activation to lipid metabolism and peroxide formation in human leucocytes. Br J Pharmacol 141: 223–232 [DOI] [PMC free article] [PubMed] [Google Scholar]
2. Appella E, Anderson CW (2001) Post-translational modifications and activation of p53 by genotoxic stresses. Eur J Biochem 268: 2764–2772 [DOI] [PubMed] [Google Scholar]
3. Atamna H, Paler-Martinez A, Ames BN (2000) N-t-butyl hydroxylamine, a hydrolysis product of alpha-phenyl-N-t-butyl nitrone, is more potent in delaying senescence in human lung fibroblasts. J Biol Chem 275: 6741–6748 [DOI] [PubMed] [Google Scholar]
4. Bischof O, Kirsh O, Pearson M, Itahana K, Pelicci PG, Dejean A (2002) Deconstructing PML-induced premature senescence. EMBO J 21: 3358–3369 [DOI] [PMC free article] [PubMed] [Google Scholar]
5. Bringold F, Serrano M (2000) Tumor suppressors and oncogenes in cellular senescence. Exp Gerontol 35: 317–329 [DOI] [PubMed] [Google Scholar]
6. Bulavin DV, Saito S, Hollander MC, Sakaguchi K, Anderson CW, Appella E, Fornace AJ (1999) Phosphorylation of human p53 by p38 kinase coordinates N-terminal phosphorylation and apoptosis in response to UV radiation. EMBO J 18: 6845–6854 [DOI] [PMC free article] [PubMed] [Google Scholar]
7. Campisi J (2001) Cellular senescence as a tumor-suppressor mechanism. Trends Cell Biol 11: 27–31 [DOI] [PubMed] [Google Scholar]
8. Catalano A, Caprari P, Soddu S, Procopio A, Romano M (2004) 5-Lipoxygenase antagonizes genotoxic stress-induced apoptotis by perturbing p53 nuclear trafficking. FASEB J 18: 1740–1742 [DOI] [PubMed] [Google Scholar]
9. Chen XS, Sheller JR, Johnson EN, Funk CD (1994) Role of leukotrienes revealed by targeted disruption of the 5-lipoxygenase gene. Nature 372: 179–182 [DOI] [PubMed] [Google Scholar]
10. Ding XZ, Tong WG, Adrian TE (2003) Multiple signal pathways are involved in the mitogenic effect of 5(S)-HETE in human pancreatic cancer. Oncology 65: 285–294 [DOI] [PubMed] [Google Scholar]
11. D'Orazi G, Cecchinelli B, Bruno T, Manni I, Higashimoto Y, Saito S, Gostissa M, Coen S, Marchetti A, Del Sal G, Piaggio G, Fanciulli M, Appella E, Soddu S (2002) Homeodomain-interacting protein kinase-2 phosphorylates p53 at Ser 46 and mediates apoptosis. Nat Cell Biol 4: 11–19 [DOI] [PubMed] [Google Scholar]
12. Drayton S, Peters G (2002) Immortalization and transformation revisited. Curr Opin Genet Dev 12: 98–104 [DOI] [PubMed] [Google Scholar]
13. Ferbeyre G, de Stanchina E, Querido E, Baptiste N, Prives C, Lowe SW (2000) PML is induced by oncogenic ras and promotes premature senescence. Genes Dev 14: 2015–2027 [PMC free article] [PubMed] [Google Scholar]
14. Forsyth NR, Evans AP, Shay JW, Wright WE (2003) Developmental differences in the immortalization of lung fibroblasts by telomerase. Aging Cell 2: 235–243 [DOI] [PubMed] [Google Scholar]
15. Fotedar R, Bendjennat M, Fotedar A (2004) Role of p21WAF1 in the cellular response to UV. Cell Cycle 3: 134–137 [PubMed] [Google Scholar]
16. Gorbunova V, Seluanov A, Pereira-Smith OM (2002) Expression of human telomerase (hTERT) does not prevent stress-induced senescence in normal human fibroblasts but protects the cells from stress-induced apoptosis and necrosis. J Biol Chem 277: 38540–38549 [DOI] [PubMed] [Google Scholar]
17. Haendeler J, Hoffmann J, Diehl JF, Vasa M, Spyridopoulos I, Zeiher AM, Dimmeler S (2004) Antioxidants inhibit nuclear export of telomerase reverse transcriptase and delay replicative senescence of endothelial cells. Circ Res 94: 768–775 [DOI] [PubMed] [Google Scholar]
18. Hong SH, Avis I, Vos MD, Martinez A, Treston AM, Mulshine JL (1999) Relationship of arachidonic acid metabolizing enzyme expression in epithelial cancer cell lines to the growth effect of selective biochemical inhibitors. Cancer Res 59: 2223–2228 [PubMed] [Google Scholar]
19. Hosni R, Chabannes B, Pacheco Y, Moliere P, Grosclaude M, Perrin Fayolle M, Lagarde M (1991) Leukotriene B4 levels from stimulated neutrophils from healthy and allergic subjects: effect of platelets and exogenous arachidonic acid. Eur J Clin Invest 21: 631–637 [DOI] [PubMed] [Google Scholar]
20. Huwiler A, Johansen B, Skarstad A, Pfeilschifter J (2001) Ceramide binds to the CaLB domain of cytosolic phospholipase A2 and facilitates its membrane docking and arachidonic acid release. FASEB J 15: 7–9 [DOI] [PubMed] [Google Scholar]
21. Jiang J, Borisenko GG, Osipov A, Martin I, Chen R, Shvedova AA, Sorokin A, Tyurina YY, Potapovich A, Tyurin VA, Graham SH, Kagan VE (2004) Arachidonic acid-induced carbon-centered radicals and phospholipid peroxidation in cyclo-oxygenase-2-transfected PC12 cells. J Neurochem 90: 1036–1049 [DOI] [PubMed] [Google Scholar]
22. Lee AC, Fenster BE, Ito H, Takeda K, Bae NS, Hirai T, Yu ZX, Ferrans VJ, Howard BH, Finkel T (1999) Ras proteins induce senescence by altering the intracellular levels of reactive oxygen species. J Biol Chem 274: 936–940 [DOI] [PubMed] [Google Scholar]
23. Lewis RA, Austen KF, Soberman RJ (1990) Leukotrienes and other products of the 5-lipoxygenase pathway. Biochemistry and relation to pathobiology in human diseases. N Engl J Med 323: 645–655 [DOI] [PubMed] [Google Scholar]
24. Los M, Schenk H, Hexel K, Baeuerle PA, Droge W, Schulze-Osthoff K (1995) IL-2 gene expression and NF-κB activation through CD28 requires reactive oxygen production by 5-lipoxygenase. EMBO J 14: 3731. [DOI] [PMC free article] [PubMed] [Google Scholar]
25. Lundberg AS, Hahn WC, Gupta P, Weinberg RA (2000) Genes involved in senescence and immortalization. Curr Opin Cell Biol 12: 705–709 [DOI] [PubMed] [Google Scholar]
26. Macip S, Igarashi M, Berggren P, Yu J, Lee SW, Aaronson SA (2003) Influence of induced reactive oxygen species in p53-mediated cell fate decisions. Mol Cell Biol 23: 8576–8585 [DOI] [PMC free article] [PubMed] [Google Scholar]
27. Martinez J, Sanchez T, Moreno JJ (2000) Regulation of prostaglandin E2 production by the superoxide radical and nitric oxide in mouse peritoneal macrophages. Free Radic Res 32: 303–311 [DOI] [PubMed] [Google Scholar]
28. Mathon NF, Lloyd AC (2001) Cell senescence and cancer. Nat Rev Cancer 1: 203–213 [DOI] [PubMed] [Google Scholar]
29. Moreno JJ (2003) Effect of olive oil minor components on oxidative stress and arachidonic acid mobilization and metabolism by macrophages RAW 264.7. Free Radic Biol Med 35: 1073–1081 [DOI] [PubMed] [Google Scholar]
30. Parrinello S, Samper E, Krtolica A, Goldstein J, Melov S, Campisi J (2003) Oxygen sensitivity severely limits the replicative lifespan of murine fibroblasts. Nat Cell Biol 5: 741–747 [DOI] [PMC free article] [PubMed] [Google Scholar]
31. Pearson M, Carbone R, Sebastiani C, Cioce M, Fagioli M, Saito S, Higashimoto Y, Appella E, Minucci S, Pandolfi PP, Pellicci PG (2000) PML regulates p53 acetylation and premature senescence induced by oncogenic Ras. Nature 406: 207–210 [DOI] [PubMed] [Google Scholar]
32. Rippo MR, Moretti S, Vescovi S, Tomasetti M, Orecchia S, Amici G, Catalano A, Procopio A (2004) FLIP overexpression inhibits death receptor-induced apoptosis in malignant mesothelial cells. Oncogene 23: 7753–7760 [DOI] [PubMed] [Google Scholar]
33. Romano M, Catalano A, Nutini M, D'Urbano E, Crescenzi C, Claria J, Libner R, Davi G, Procopio A (2001) 5-Lipoxygenase regulates malignant mesothelial cell survival: involvement of vascular endothelial growth factor. FASEB J 15: 2326–2336 [DOI] [PubMed] [Google Scholar]
34. Schmitt CA (2003) Senescence, apoptosis and therapy cutting the lifelines of cancer. Nat Rev Cancer 3: 286–295 [DOI] [PubMed] [Google Scholar]
35. Serrano M, Blasco MA (2001) Putting the stress on senescence. Curr Opin Cell Biol 13: 748–753 [DOI] [PubMed] [Google Scholar]
36. Serrano M, Lin AW, McCurrach ME, Beach D, Lowe SW (1997) Oncogenic ras provokes premature cell senescence associated with accumulation of p53 and p16INK4a. Cell 88: 593–602 [DOI] [PubMed] [Google Scholar]
37. Soberman RJ, Christmas P (2003) The organization and consequences of eicosanoid signaling. J Clin Invest 111: 1107–1113 [DOI] [PMC free article] [PubMed] [Google Scholar]
38. Stewart SA, Weinberg RA (2000) Telomerase and human tumorigenesis. Semin Cancer Biol 10: 399–406 [DOI] [PubMed] [Google Scholar]
39. Wang W, Chen JX, Liao R, Deng Q, Zhou JJ, Huang S, Sun P (2002) Sequential activation of the MEK-extracellular signal-regulated kinase and MKK3/6-p38 mitogen-activated protein kinase pathways mediates oncogenic ras-induced premature senescence. Mol Cell Biol 22: 3389–3403 [DOI] [PMC free article] [PubMed] [Google Scholar]
40. Wei S, Wei S, Sedivy JM (1999) Expression of catalytically active telomerase does not prevent premature senescence caused by overexpression of oncogenic Ha-Ras in normal human fibroblasts. Cancer Res 59: 1539–1543 [PubMed] [Google Scholar]
41. Woo CH, Eom YW, Yoo MH, You HJ, Han HJ, Song WK, Yoo YJ, Chun JS, Kim JH (2000) Tumor necrosis factor-á generates reactive oxygen species via a cytosolic phospholipase A2-linked cascade. J Biol Chem 275: 32357–32362 [DOI] [PubMed] [Google Scholar]

Supplementary Materials