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5-Lipoxygenase antagonizes genotoxic stress-induced apoptosis by altering p53 nuclear trafficking

ALFONSO CATALANO^*,1, PAOLA CAPRARI^*,, SILVIA SODDU, ANTONIO PROCOPIO^*,2 and MARIO ROMANO^,2

^* Department of Molecular Pathology and Innovative Therapies, Polytechnic University of Marche, Ancona, Italy;
Molecular Oncogenesis Laboratory, Regina Elena Cancer Institute, Rome, Italy;
Department of Biomedical Sciences and Aging Research Center, Ce. S. I., "Gabriele D’Annunzio" University Foundation, Chieti, Italy; and
Neural Development Group, Mouse Cancer Genetics Program, National Cancer Institute, Frederick, Maryland, USA

¹Correspondence: Dipartimento di Patologia Molecolare e Terapie Innovative, Università Politecnica delle Marche, Via Ranieri, Ancona 60131, Italy. E-mail: catgfp{at}yahoo.it

SPECIFIC AIMS

Arachidonate 5-lipoxygenase (5-LO) is emerging as a key regulator of cancer cell proliferation and survival. The aim of this study was to elucidate the relationship between 5-LO expression/function and molecular events regulated by the tumor suppressor p53 in response to genotoxic stress. Our goal was to determine whether 5-LO could be involved in cancer chemoresistance.

PRINCIPAL FINDINGS

1. 5-LO expression and activity are up-regulated by genotoxic agents
Genotoxic agents, such as H₂O₂ (100 µM), etoposide (10 µM), and adriamycin (ADR, 250 ng/mL), as well as (TNF) (50 ng/mL), but not Fas ligand (FasL, 100 ng/mL), induced a time-dependent expression of 5-LO protein in MPP89 mesothelioma cells (Fig. 1 A) with a maximum reached at 16 h in the case of ADR (Fig. 1B ). This agent also stimulated 5-LO mRNA accumulation (Fig. 1C ), 5-LO translocation to the nuclear envelope (Fig. 1D ), and 5-HETE accumulation in conditioned medium (Fig. 1E ). ADR potently enhanced 5-LO protein expression in glioma U87GM (wild-type p53), breast cancer MCF7 (wild-type p53), prostate cancer PC3 (p53 null), and colon cancer CCL-238 (p53 null) cell lines (Fig. 1F ), suggesting that 5-LO up-regulation by genotoxic stress is not restricted to mesothelioma cells and is independent of p53.

View larger version (37K): [in this window] [in a new window]   Figure 1. Genotoxic stress stimulates 5-LO expression, nuclear translocation and activity. A) Western blot analysis of 5-LO protein in MPP89 cells exposed to the indicated agents for 4 or 16 h. B) Time course of 5-LO protein expression in ADR (250 ng/mL)- () or vehicle-treated () MPP89 cells. Data points derive from scanning densitometry and are normalized for -tubulin expression. Results are the mean ± SE of n = 3. C) RT-PCR semiquantitation of 5-LO mRNA levels in MPP89 cells exposed to ADR (250 ng/mL) for the indicated time. D) 5-LO immunostaining (green) in MPP89 cells either left untreated (vehicle control) or treated with ADR (250 ng/mL, 16 h). DAPI staining of the nuclei (blue) was carried out in parallel. E) RP-HPLC measurements of 5-HETE in media conditioned by MPP89 cells for 48 h. Results represent the mean ± SE of n = 3. *P 0.05 compared with control. F) Western blot analysis of 5-LO protein expression in the indicated cell lines exposed or not to ADR (250 ng/mL) for 16 h. Expression of -tubulin was used as internal control.

2. 5-LO reduces genotoxic-stress induced apoptosis
To assess the functional role of 5-LO in cancer cell response to genotoxic damage, we stably transfected the human lung cancer cell line A549 (5-LO negative; wild-type p53) with wild-type 5-LO-GFP fusion protein (WT-5LO clones) or catalytically inactive 5-LO mutant, H367Q-GFP fusion protein (Mut-5LO clones). When exposed for 24 h to 25 or 250 ng/mL of ADR, WT-5LO clones exhibited significantly lower nucleosome formation compared with parental cells or Mut-5LO clones. These results were confirmed by cytofluorimetric analysis of the cell cycle. Upon exposure to ADR (250 ng/mL) the percent of cells in sub-G1 was 16.9 in WT-5LO clones, 51.4 in Mut-5LO, and 52.6 in parental A549 cells. AA-861 (1 µM), a selective 5-LO inhibitor, abrogated resistance of WT-5LO clones as well as of 5-LO-expressing MPP89 cells to ADR-induced apoptosis. On the other hand, 5(S)-HETE concentration dependently reduced nucleosome formation induced by ADR in A549 cells.

3. 5-LO selectively suppresses p53-dependent transcription of proapoptotic genes
p53 activation is the main pathway of genotoxic stress-induced apoptosis. Thus, we examined whether 5-LO may impair p53 functions. We transiently transfected A549, WT-5LO, and Mut-5LO cells with a p53 cDNA and evaluated the apoptotic response. After transfection, p53 protein was equally expressed in the three clones. We denoted a significant formation of nucleosomes in parental A549 and Mut-5LO cells, but not in WT-5LO cells. In contrast, FasL (100 ng/mL, 24 h) induced a strong apoptotic response in A549 cells as well as in Mut-5LO or WT-5LO cells.

To investigate the mechanisms of 5-LO inhibition of p53-dependent apoptosis, we analyzed the expression pattern of p53-transactivated proteins in A549, Mut-5LO, and WT-5LO cells treated with ADR (250 ng/mL) for varying times. p53, MDM-2 (p53 inhibitor), and p21 (cell cycle arrest-related protein), timely increased in both Mut-5LO and WT-5LO cells as well as in A549 parental cells. In contrast, expression of pro-apoptotic proteins BAX and PIG3 timely decreased in WT-5LO cells, but not in Mut-5LO cells. AA-861 (1 µM) consistently increased BAX levels in MPP89 cells treated with ADR without altering p53 or p21 expression. This effect was abrogated by 5(S)-HETE (100 nM). 5-LO regulation of p53-induced proteins occurs at the transcriptional level. Cotransfection of the pCMV-p53 vector (100 ng) with constructs (0.5 µg) expressing the promoter regions of p53-inducible genes fused to a luciferase reporter (luc), activated bax-luc in A549 cells, and Mut-5LO cells, but not in WT-5LO cells, whereas it activated p21-luc at a similar extent in the A549 parental cells and two sister clones. AA-861 clearly up-regulated bax-luc activity in WT-5LO cells, but not in Mut-5LO cells treated with ADR. 5(S)-HETE significantly reduced bax-luc activity in both cell types. The p53 dependence of these phenomena was confirmed by the inability of ADR, alone or in combination with AA-861 or 5(S)-HETE, to activate a mutated bax construct, which does not bind p53.

4. 5-LO alters p53 relocalization in NBs and antagonizes p53/PML physical association
To investigate the mechanisms of 5-LO actions on p53 functions, we examined p53 intracellular localization using indirect immunofluorescence and confocal laser microscopy. Upon exposure to ADR, Mut-5LO cells as well as A549 parental cells displayed nuclear speckles of PML fluorescence. p53 fluorescence also appeared as punctuate foci, which in 15% of cases coincided with PML immunostaining. By contrast, in nuclei of WT-5LO cells, p53 immunofluorescence appeared as large blocks, clearly distinct from PML speckles, which instead showed a similar pattern as that of Mut-5LO cells. AA-861 (1 µM) restored p53 localization into PML-NBs in WT-5LO cells, whereas 5(S)-HETE (100 nM) profoundly altered the nuclear distribution pattern of p53 in A549 cells, giving diffuse nuclear fluorescence, although it did not change PML immunostaining. When we exposed the mesothelioma cells MPP89 to ADR (250 ng/mL), we consistently observed a speckle distribution of PML in a large majority of cells (up to 90%). p53 fluorescence appeared as large nuclear dots that only occasionally (6% of cases) localized together with PML-NBs. AA-861 gave a marked redistribution of p53 immunostaining, that in 20% of cases colocalized with PML. This effect was reversed by 5(S)-HETE. To obtain a more direct evidence of 5-LO perturbation of p53/PML association, we immunoprecipitated lysates from A549, Mut-5LO, and WT-5LO cells transfected with p53 and exposed for 4 h to As₂O₃ (1 µM), an agent known to up-regulate PML. We used a specific anti-PML antibody (PG-M3) for immunoprecipitation and immunoblotted the precipitates with the anti-p53 antibody, DO-1. As expected, p53 coimmunoprecipitated with PML in A549 or Mut-5LO cells, but not in WT-5LO cells (Fig. 2 A, lanes 1, 2). AA-861, which was ineffective with A549 and Mut-5LO cells, increased p53/PML association in WT-5LO cells (Fig. 2A , lanes 3, 4). Conversely, 5(S)-HETE suppressed p53/PML coimmunoprecipitation in A549 cells (Fig. 2B ). Thus, 5-LO activity selectively impairs p53-induced apoptosis by altering p53/PML association. A diagrammatic representation of our findings is reported in Fig. 3 .

View larger version (39K): [in this window] [in a new window]   Figure 2. 5-LO inhibits p53/PML association. A549, Mut-5LO, and WT-5LO cells were transfected with a p53 cDNA. A) 24 h after transfection cells were incubated with either As2O3 (1 µM) or AA-861 (1 µM) for 4 h. Cells were lysed and proteins immunoprecipitated (IP) using an anti-PML antibody (PG-M3). Immunoprecipitates were analyzed by Western blot (WB) with anti-p53 antibody (DO-1). The amount of PML in the immune complex was determined by reprobing the same membrane with anti-PML. Expression levels of p53 and PML after transfection and treatment was determined using anti-p53 or anti-PML antibodies. B) Lysates from cells treated with either As2O3 (1 µM) or 5(S)-HETE (100 nM) for 4 h and precipitated with anti-PML antibody (PG-M3) were analyzed by Western blot with an anti-p53 antibody.

View larger version (33K): [in this window] [in a new window]   Figure 3. Schematic diagram.

CONCLUSIONS AND SIGNIFICANCE

Induction of cancer cell apoptosis by genotoxic damage involves up-regulation of tumor suppressor p53, which initiates transcription of apoptotic genes. Genetic or epigenetic disruption of p53 pathways is associated with chemoresistance. We found that 5-LO expression and activity are stimulated by genotoxic agents and that these events may carry functional consequences. We observed that: 1) 5-LO overexpression confers resistance to ADR-induced apoptosis; 2) this resistance is entirely dependent on 5-LO catalytic activity, since it was not observed in Mut-5LO cells and was fully reproduced by 5-LO metabolite 5(S)-HETE; and 3) suppression of constitutive 5-LO strongly increases chemosensitivity of cancer cells. These findings point to 5-LO as an endogenous regulator of cancer cell chemosensitivity, suggesting that 5-LO inhibitors could be used in adjunction with genotoxic drugs to overcome chemoresistance.

The mechanisms of p53 inhibition by 5-LO appear to be dependent on the capability of 5-LO to alter p53 nuclear distribution. PML binds to the core domain of p53, contributing to the localization of p53 in NBs. Within this subnuclear region, transcriptional activation of proapoptotic genes (such as bax and pig3) is selectively enhanced. How this is achieved is incompletely defined. We provide evidence indicating that 5-LO activity inhibits the physical interaction between p53 and PML, thereby altering the pattern of p53 subnuclear distribution in response to genotoxic stress. To the best of our knowledge, arachidonic acid metabolism by 5-LO represents the first recognized signaling event that regulates p53 targeting to PML-NBs and, as a consequence, that selectively inhibits p53 proapoptotic functions in cancer cells. This finding may explain, at least in part, the paradox of cancer cells that express a wild-type p53, but are chemoresistant.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-2258fje;

² These authors share the senior authorship of this work.

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