Oncogene (2006) 25, 2203–2212 & 2006 Nature Publishing Group All rights reserved 0950-9232/06 $30.00 www.nature.com/onc ORIGINAL ARTICLE Regulation of p53 and suppression of apoptosis by the soluble guanylyl cyclase/cGMP pathway in human ovarian cancer cells

M Fraser1,2,3, SL Chan1,2,3,4, SSL Chan1,2,3, RR Fiscus4 and BK Tsang1,2,3

1Reproductive Biology Unit and Division of Gynecologic Oncology, Department of Obstetrics & Gynecology, University of Ottawa, Ottawa, Ontario, Canada; 2Department of Cellular & Molecular Medicine, University of Ottawa, Ottawa, Ontario, Canada; 3Hormones, Growth and Development Program, Ottawa Health Research Institute, The Ottawa Hospital (Civic Campus), Ottawa, Ontario, Canada and 4Department of Physiology (Faculty of Medicine), Epithelial Cell Biology Research Centre and Centre for Gerontology and Geriatrics, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong

Dysregulated apoptosis plays a critical role in the Oncogene (2006) 25, 2203–2212. doi:10.1038/sj.onc.1209251; development of a number of aberrant cellular processes, published online 14 November 2005 including tumorigenesis and chemoresistance. However, the mechanisms that govern the normal apoptotic program Keywords: ovarian cancer; apoptosis; soluble guanylyl are not completely understood. Soluble guanylyl cyclase cyclase; cGMP; p53 (sGC) and cyclic guanosine monophosphate (cGMP) promote mammalian cell viability via an unknown mechanism and p53 status is a key determinant of cell fate in human ovarian cancer cells. Whether an interaction exists between these two determinants of cell fate is Introduction unknown. We hypothesized that basal sGC activity reduces p53 content and attenuates p53-dependent apop- Dysregulated apoptosis contributes to the development tosis in human ovarian cancer cells. Suppression of sGC of a number of pathologies, including cancer (Lieber- activity with the specific inhibitor 1H-[1,2,4]oxadiazo- mann et al., 1995; Reap et al., 1995; Estevez et al., 1998; lo[4,3-a]quinoxalin-1-one (ODQ) lowered cGMP content, Fiscus, 2002; Fiscus et al., 2002). Tumor growth results, and increased p53 protein content and induced apoptosis in part, from an imbalance between cell proliferation in three ovarian cancer cell lines, effects which were and apoptosis (Kerr et al., 1994; Levine et al., 1995; attenuated by the cGMP analog 8-Br-cGMP and by Sheets and Yeh, 1997; LaCasse et al., 1998) and our Atrial Natriuretic Factor, an activator of particulate laboratory has been interested in the role of dysregu- guanylyl cyclase, which circumvent the inhibition of sGC. lated apoptosis in the regulation of cell fate in human ODQ prolonged p53 half-life, induced phosphorylation of ovarian cancer cells (Sasaki et al., 2000; Asselin et al., p53 on Ser15, and upregulated the p53-dependent gene 2001; Fraser et al., 2003). products p21, murine double minute-2, and the proapop- Soluble guanylyl cyclase (sGC) is one of the major totic, p53-responsive gene product Bax. ODQ activated producers of basal cyclic guanosine monophosphate caspase-3, and ODQ-induced apoptosis was inhibited by (cGMP) content in mammalian cells (Waldman and overexpression of X-linked inhibitor of apoptosis Protein. Murad, 1987; Garthwaite et al., 1995; Fiscus, 2002), and Pretreatment with the specific p53 inhibitor pifithrin or is necessary for the survival of several cell types under downregulation of p53 using a specific small inhibitory normal growth conditions, as well as for protection RNA significantly attenuated ODQ-induced apoptosis. against various apoptotic stimuli (Flamigni et al., 2001). Moreover, ODQ-induced upregulation of p21 and Bax Our previous studies have demonstrated that the basal and ODQ-induced apoptosis were significantly reduced in activities of sGC and a downstream target protein in the a p53 mutant cell line relative to the wild-type parental cGMP signalling pathway, protein kinase G (PKG), cell line. Thus, the current study establishes that basal may be important for the protection of uterine epithelial sGC/cGMP activity regulates p53 protein stability, cells (Chan and Fiscus, 2003) and neural cell lines content, and function, possibly by altering p53 phosphor- (Fiscus, 2002) against spontaneous apoptosis. However, ylation and stabilization, and promotes cell survival in the precise mechanisms by which sGC/cGMP regulates part through regulation of caspase-3 and p53. cell fate are not known. While p53 status is a determinant of cisplatin resistance in human ovarian cancer cells and failure to properly regulate p53 may be a contributing factor Correspondence: Dr BK Tsang, Ottawa Health Research Institute, 725 tochemoresistance(Fraser et al., 2003), precisely how Parkdale Avenue, Ottawa, Ontario, Canada K1Y 4E9. E-mail: [email protected] p53 is regulated in these cells is unclear. In the current Received 9 August 2005; revised 7 October 2005; accepted 7 October study, the possibility that the constitutive presence 2005; published online 14 November 2005 of sGC-derived cGMP is an important negative cGMP regulates p53 and apoptosis in ovarian cancer cells M Fraser et al 2204 regulator of p53 content and p53-dependent apop- Results tosis in human ovarian cancer cells is examined. The current study demonstrates that basal sGC-derived ODQ (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one) cGMP is required for the viability of human ovarian depletes cellular cGMP levels cancer cells, likely by inhibiting the activation of To determine whether sGC is an important regulator of execution caspases such as caspase-3. Furthermore, cGMP content in human ovarian cancer cells, OV2008 these findings suggest that sGC/cGMP-mediated survi- cells were cultured for 48 h, and then treated 20 min with val of these cells proceeds, in part, by lowering basal p53 ODQ (0–100 mM), a potent and specific sGC inhibitor protein content and phosphorylation and p53-induced (Garthwaite et al., 1995; Moro et al., 1996; Mulsch apoptosis, and that the basal activity of this pathway et al., 1997; Olesen et al., 1998). Cells were harvested may alter p53 content and function by promoting p53 and cGMP assays were performed as detailed in degradation. Materials and methods. As shown in Figure 1a, ODQ

Figure 1 Depletion of cGMP and induction of apoptosis by the sGC inhibitor ODQ in human ovarian cancer cells. (a) OV2008 cells were cultured for 48 h and then treated with ODQ (0–100 mM) for 20 min. cGMP assays were performed as detailed in Materials and methods. Histogram shows mean7s.e.m. of five independent experiments. ODQ significantly reduced cGMP content overall (Po0.0001). Moreover, at 50 mM ODQ, cGMP content was reduced by >60%, compared to control (Po0.001), while at 100 mM ODQ, cGMP content was B70% lower than control cells (Po0.001). Results demonstrate that inhibition of sGC activity lowers cGMP levels, suggesting that sGC is a major regulator of basal cGMP levels in ovarian cancer cells. *Po0.05, **Po0.001, relative to untreated control cells. (b) OV2008 and A2780s human ovarian cancer cells were cultured in the presence of the soluble guanylyl cyclase (sGC) inhibitor ODQ (0–50 mM), in the presence or absence of the stable cGMP analog 8-Br-cGMP (1 mM; top panel). Histograms show mean7s.e.m. of four independent experiments. ODQ significantly increased the number of apoptotic cells in both OV2008 and A2780s cells (Po0.0001, both cell lines). Moreover, 8-Br-cGMP significantly attenuated the effects of ODQ (Po0.01, OV2008; Po0.05, A2780 s). (c) A2780s cells were cultured for 24 h in the presence of ODQ (0, 30 mM). Floating and attached cells were pooled and apoptosis was determined by TUNEL assay as described in Materials and methods. (d) A2780s cells were treated with ODQ (0, 30 mM) in the presence of Atrial Natriuretic Factor (ANF, 0–1 mM; bottom panel), an activator of particulate guanylyl cyclase. Histograms show mean7s.e.m. of five independent experiments. ANF significantly attenuated ODQ-induced apoptosis (Po0.01). Results suggest that sGC is a major regulator of basal cGMP content in ovarian cancer cells, that sGC activity is required for basal viability of human ovarian cancer cells, and that the effects of ODQ are likely mediated through the cGMP pathway. ***Po0.001, relative toequimolarODQ, 8-Br-cGMP-treated cells; ** Po0.01, relative toequimolar, 8-Br-cGMP-treated cells.

Oncogene cGMP regulates p53 and apoptosis in ovarian cancer cells M Fraser et al 2205 significantly lowered cellular cGMP levels in a concen- tration-dependent manner (Po0.0001). Moreover, at 50 and 100 mM, ODQ lowered cGMP content by 63 and 70%, respectively (Po0.001, relative tountreated control). Even at 30 mM, ODQ significantly reduced cGMP levels, relative to control (Po0.05). These results demonstrate that inhibition of sGC activity lowers cGMP levels and suggest that sGC is a major regulator of basal cGMP levels in ovarian cancer cells.

Induction of apoptosis by the sGC inhibitor ODQ ODQ induces apoptosis in immortalized uterine epithe- lial cells (Chan and Fiscus, 2003) as well as in established neural cell lines (Fiscus, 2002), suggesting that basal levels of sGC activity are important for preventing spontaneous induction of apoptosis. How- ever, it is unclear whether this pathway affects the viability of human ovarian cancer cells, where disrup- tions of apoptotic mediators is commonly observed. Toaddress this question,A2780s and OV2008 human ovarian cancer cell lines were incubated for 24 h in the presence or absence of ODQ (0, 30, 50 mM). DMSO was used as a control and maintained at a concentration of 0.1% (v/v) in all groups. Previous studies demonstrated Figure 2 ODQ-induced apoptosis is associated with activation of that ODQ at 40 mM completely inhibited sGC activity, caspase-3 and is inhibited by Xiap. (a) A2780s and OV2008 cells were cultured for 24 h in the presence of ODQ (0, 30, 50 mM). p17 lowered basal levels of cGMP to one-fifth of normal active caspase-3 content in whole-cell lysates was determined by levels and induced apoptosis in neural cell lines (Fiscus, Western blotting. Blots are representative of three independent 2002). In the present study, exposure of A2780s or experiments. ODQ markedly upregulated the active form of OV2008 cells to ODQ caused a concentration-dependent caspase-3 in a concentration-dependent manner in both cell lines. B increase in apoptosis, as measured by Hoechst nuclear (b) A2780s cells were cultured for 18 h, and then infected with adenovirus containing cDNA for Xiap or LacZ (MOI ¼ 0, 2.5, 5, staining (Po0.0001; Figure 1b). ODQ-induced apopto- 10). Following 24 h of infection, the virus was removed and the sis was alsoconfirmed by TUNEL assay. As expected, cells were treated with ODQ (0, 100 mM) for a further 24 h. ODQ (30 mM) increased the percentage of TUNEL- Overexpression of Xiap was confirmed by Western blot analysis positive (i.e. apoptotic) A2780s cells (Po0.0001), (representative of three independent experiments). ODQ signifi- 7 7 cantly induced apoptosis (Po0.001), relative tothe untreated relative to DMSO control (20 1.25% vs. 0.7 0.15%; control. However, these effects were inhibited by overexpression of Figure 1c). The proapoptotic effects of ODQ were Xiap in a concentration-dependent manner. ***Po0.001, relative significantly attenuated by the cell-permeable, stable toCTL; * Po0.05, **Po0.01, relative tovirus-free, ODQ-treated cGMP analog 8-Br-cGMP (OV2008, Po0.01; A2780 s, cells. Po0.05). Furthermore, ODQ-induced apoptosis was significantly attenuated by the particulate guanylyl cyclase (pGC) activator Atrial Natriuretic Factor X-linked inhibitor of apoptosis protein (Xiap) is a (ANF; Figure 1d, P 0.001, all effects), suggesting that o potent endogenous inhibitor of caspase-3, -7, and -9, sGC activity is required for the viability of ovarian and is a central regulator of cell fate in human ovarian cancer cells, and that ODQ-induced apoptosis is specific cancer cells (Sasaki et al., 2000; Asselin et al., 2001; to its inhibitory effects on sGC. Li et al., 2001). Todetermine whether ODQ-induced apoptosis is similarly regulated, Xiap was overexpressed ODQ activates caspase-3 and induces X-linked inhibitor using an adenoviral sense Xiap cDNA (Xiap-s). Cells of apoptosis protein (Xiap)-sensitive apoptosis were infected for 24 h with Xiap-s (MOI ¼ 0, 2.5, 5, 10; Since activation of the caspase cascade is a critical LacZ control), and then treated with ODQ (0, 100 mM) event for the induction of apoptosis in mammalian for a further 24 h. As shown in Figure 2b, ODQ cells, whether inactivation of the sGC/cGMP pathway significantly increased the percentage of apoptotic cells, is associated with changes in caspase-3 activity were relative tothe untreated cells ( Po0.001). However, next determined. A2780s and OV2008 cells were treated while infection with the LacZ adenovirus had no effect with ODQ (0, 30, 50 mM) for 24 h and p17 (active) on ODQ sensitivity, Xiap-s inhibited ODQ-induced caspase-3 content was determined by Western blotting. apoptosis in a concentration-dependent manner As shown in Figure 2a, ODQ markedly upregulated the (Po0.01). Overexpression of Xiap was confirmed by active form of caspase-3 in a concentration-dependent Western blot analysis (Figure 2b, lanes 3, 4, and 5, manner in both cell lines, suggesting that suppression of compared to lane 1). Interestingly, ODQ alone down- basal sGC activity may result in increased caspase-3 regulated Xiap (lane 2 vs lane 1), suggesting that activity. regulation of Xiap may be involved in ODQ-induced

Oncogene cGMP regulates p53 and apoptosis in ovarian cancer cells M Fraser et al 2206 apoptosis. These results demonstrate that ODQ-induced apoptosis is inhibited by Xiap, and suggest that ODQ- induced apoptosis involves modulation of a Xiap- regulated pathway, such as caspase-3 activation.

p53 is regulated by the sGC/cGMP pathway and is required for maximal ODQ-induced apoptosis Since p53 is an important regulator of cell fate in human ovarian cancer cells, we next examined whether ODQ- induced apoptosis is associated with changes in p53 content in these cells. A2780s, OV2008, and C13* cell lines were treated with ODQ as above and p53 contents were determined by Western blotting. ODQ markedly upregulated p53 in all three cell lines (Figure 3a), suggesting that p53 may be involved in apoptosis induced by sGC inhibition. Furthermore, while ODQ upregulated p53 in both A2780s and OV2008 cells (30 and 50 mM, respectively), this was significantly attenuated by 8-Br-cGMP (1 mM; Figure 3b, Po0.01), suggesting that ODQ-induced upregulation of p53 and induction of apoptosis is secondary to suppression of cellular cGMP levels. 8- Br-cGMP did not have a measurable effect on basal p53 levels, suggesting that in unstressed cells, p53 levels are kept at their lowest possible levels. Todetermine whether p53 is required forODQ- induced apoptosis, we treated A2780s cells with ODQ (30 mM; 24 h) in the presence of the specific p53 inhibitor pifithrin-a hydrobromide (PFT) (0–30 mM). PFT is an inhibitor of p53 transactivation and p53-dependent apoptosis, which may function by preventing the nuclear import of p53 following cell stress (Komarov et al., 1999). This inhibitor markedly attenuates the p53- induced expression of p21WAF1/CIP1, and its biological effects are reversible by overexpression of p53 (Fraser Figure 3 Upregulation of p53 protein content through inhibition et al., 2003). As shown in Figure 4a, while ODQ of the sGC/cGMP pathway. (a) A2780s, OV2008, and C13* cells significantly increased the number of apoptotic cells were cultured for 24 h in the presence of ODQ (0, 30, 50 mM). p53 (P 0.0001), PFT significantly, although not comple- protein content was assessed by Western blot analysis using an o anti-p53 antibody (A2780s – clone 80; OV2008 – clone DO-1). tely, attenuated ODQ-induced apoptosis (Po0.01), Results were normalized for protein loading by reprobing the blots suggesting that p53 function is, at least in part, required with an anti-GAPDH antibody. Western blots are representative of for ODQ-induced apoptosis. However, even at 30 mM three independent experiments. ODQ markedly upregulated p53 in PFT, ODQ was able toinduce significant apoptosis, a concentration-dependent manner in all cell lines. (b) A2780s (top panel) or OV2008 (bottom panel) cells were cultured for 24 h in the M suggesting either that 30 m PFT does not completely presence of ODQ (0, 30, or 50 mM, as indicated) in the presence of inhibit the proapoptotic effects of p53, or that ODQ 8-Br-cGMP (0, 1 mM). p53 protein content was assessed by may induce both p53-dependent and -independent Western blot analysis. Protein content was determined by apoptosis. Furthermore, while the inhibitory effects of densitometry and is shown as mean7s.e.m. of three independent PFT on p53-mediated gene transcription are well experiments. ODQ significantly increased p53 content in A2780s cells by over fourfold and in OV2008 cells by greater than twofold, documented, it is unclear whether PFT inhibits the relative to untreated control (***Po0.001, **Po0.01). These putative mitochondrial effects of p53 (Mihara et al., effects were significantly attenuated by 8-Br-cGMP (Po0.05). 2003). Thus, to further examine the role of p53 in sGC- Results suggest that p53 protein content is regulated by the sGC/ mediated cell survival, we downregulated p53 by RNA cGMP pathway. **Po0.01, relative toequimolarODQ, CTL- interference (RNAi) in ODQ-treated A2780s cells using treated cells. a p53-specific small inhibitory RNA (siRNA). The cells were transfected for 24 h, and then treated with ODQ (0, 30 mM) for a further 24 h. The p53 siRNA (50 nM) markedly lowered p53 protein content in the absence (Po0.05). Together, these data suggest that upregula- and presence of ODQ (Figure 4b), relative to the control tion of p53 is, at least in part, required for ODQ-induced siRNA. Furthermore, while ODQ significantly induced apoptosis and that the sGC/cGMP pathway contributes apoptosis (Po0.0001), this effect was significantly, to cell survival by promoting a reduction of basal p53 although not completely, inhibited by the p53 siRNA content.

Oncogene cGMP regulates p53 and apoptosis in ovarian cancer cells M Fraser et al 2207 p53 mutant cells show reduced response to ODQ-induced domain (Olivier et al., 2002) (p53 mutation database, apoptosis version R8, June 2003). While A2780cp cells expressed Since mutation of the TP53 gene is a frequent more p53 protein in the absence of ODQ than did the occurrence in human ovarian cancer (Havrilesky et al., A2780s cells (Figure 4c, lane 1 vs. lane 4), ODQ only 2003; Leitao et al., 2004), whether p53 mutation affects slightly upregulated p53 in the A2780cp cells while p53 the requirement for sGC activity for basal survival of was markedly upregulated by ODQ in A2780s cells. ovarian cancer cells was next determined. To accomplish Furthermore, while ODQ upregulated the p53-respon- this, A2780s cells (wt p53) and the p53-mutant counter- sive gene products p21WAF1/CIP1 and Bax in a concentra- part cell line, A2780cp, were cultured in the presence of tion-dependent manner in the A2780s cells, these ODQ (as above). The passage number of both cell lines responses were markedly attenuated in A2780cp cells, was equal during cotreatment, thereby eliminating the despite the high p53 content in these cells (Figure 4c). confounding effects of passage across cell lines. A2780cp This suggests that p53 function is markedly attenuated cells express p53 mutated in helix 1 of the DNA-binding in the p53 mutant A2780cp cell line. Moreover, ODQ- induced apoptosis was significantly attenuated in A2780cp cells, relative toA2780s cells ( Po0.01), suggesting that modulation of p53-mediated apoptosis is one important mechanism by which the sGC/cGMP pathway regulates cell fate.

ODQ increases p53 stability and induces its phosphorylation on Ser 15 The mechanism by which the sGC/cGMP pathway contributes to low basal p53 content and p53-mediated apoptosis is unclear. Since p53 can be regulated by murine double minute-2 (MDM2)-mediated ubiquitina- tion and proteasomal degradation, we next wanted to determine whether inhibition of sGC affects p53 stability. Tothat end, A2780s cells were ‘pulsed’ with

Figure 4 The sGC/cGMP pathway regulates cell viability, in part, through activation of p53. (a) A2780s cells were cultured for 24 h in the presence of ODQ (0, 30 mM) in the absence or presence of the specific p53 inhibitor pifithrin-a hydrobromide (PFT; 0–30 mM). Results shown as mean7s.e.m. of three independent experiments. ODQ significantly increased the number of apoptotic cells (Po0.0001). This effect was significantly, although not completely, attenuated by PFT (Po0.01). ***Po0.001, relative toequimolar PFT, ODQ-treated cells; **Po0.01, relative toequimolarPFT, ODQ-treated cells. (b) A2780s cells were transfected with a p53- specific siRNA (0, 50 nM; scrambled sequence as control) for 24 h, and subsequently treated for a further 24 h with ODQ (0, 30 mM). Protein content and extent of apoptosis were assessed as indicated above. Western blot is representative, and histogram shows mean7s.e.m., of three independent experiments. ODQ significantly increased the number of apoptotic cells (Po0.0001). This was significantly, although not completely, attenuated by downregula- tion of p53 using the p53 siRNA (Po0.05). (c) A2780s cells and the p53 mutant variant cell line A2780cp were cultured for 24 h in the presence of ODQ (0, 15, 30 mM). Histogram shows mean7s.e.m. of three independent experiments. ODQ significantly increased the number of apoptotic cells in both cell lines (Po0.0001). However, apoptosis was significantly, although not completely, attenuated in the p53 mutant A2780cp cell line, relative toits wild-type p53 counterpart, A2780s (Po0.0001). To confirm the lack of p53 function in the A2780cp cells, we tracked p53, Bax, and p21WAF1/CIP1 content by Western blot analysis (representative of three indepen- dent experiments). ODQ markedly upregulated p53, p21, and Bax content in the wild-type A2780s cells. Despite the high levels of (mutant) p53 in the A2780cp cells, only minimal p21 and Bax immunoreactivity was observed in this cell line. Taken together, these data suggest that the upregulation of p53 plays a role in ODQ-induced apoptosis, and that the sGC/cGMP pathway regulates basal cell viability, at least in part, through down- regulation of p53. ***Po0.001, relative toCTL0transfected, CTL- treated cells; *Po0.05, relative top53-transfected, CTL-treated cells.

Oncogene cGMP regulates p53 and apoptosis in ovarian cancer cells M Fraser et al 2208 ODQ (30 mM; DMSO control) for 6 h, followed by a regulation of human cancer cell viability. Furthermore, ‘chase’ period (0–240 min) in the presence of 10 mg/ml our data represent, to our knowledge, the first report cycloheximide (CHX) (see Materials and methods). This that directly demonstrates that this pathway influences concentration of CHX was chosen based on previous mammalian cell viability by regulating the stability and studies performed in this cell line (Xiao et al., 2003). activation of p53 and p53-mediated apoptosis. Whole-cell lysates were analysed for p53 content by sGC is activated by the binding of nitric oxide (NO) Western blot. Blots were exposed such that p53 content toits heme moiety(Waldman and Murad, 1987). sGC in each treatment group (i.e. ODQ vs DMSO) was also has basal activity and contributes to most of the determined within its linear range to avoid the possible basal cGMP levels found in mammalian cells (Waldman confounding effects of ODQ-mediated p53 upregulation and Murad, 1987; Garthwaite et al., 1995; Fiscus, 2002). over the ‘pulse’ period. As shown in Figure 5a, in the While activation of sGC and elevation of cGMP levels presence of DMSO, p53 was reduced to less than half of inhibits apoptosis in many cell types (Kim et al., 1999; its time zerocontentin o30 min post-CHX. By contrast, Fiscus et al., 2001; Fiscus, 2002; Fiscus et al., 2002), ODQ markedly extended p53 half-life to120–240 min, inhibition of basal sGC activity with specific inhibitors suggesting a 4–8-fold increase in p53 half-life. (e.g. ODQ) lowers basal cGMP levels and induces While MDM2 is a key negative regulator of p53 apoptosis (Garthwaite et al., 1995; Flamigni et al., 2001; stability, it is also a p53-responsive gene product. Fiscus, 2002; Chan and Fiscus, 2003). ODQ markedly Furthermore, the interaction of MDM2 with p53 is increases caspase activity, mitochondrial cytochrome c attenuated by phosphorylation of p53 on Ser15. For release, reduces Bcl-2 content and Bad dephosphoryla- these reasons, an analysis of MDM2 levels may not be tion and cell viability (Flamigni et al., 2001). The present indicative of the role of MDM2 in p53 stabilization. To study demonstrates that basal sGC activity/cGMP that end, we next examined the effects of ODQ on content is required for viability in cultured ovarian MDM2 content and p53 Ser15 phosphorylation. A2780s cancer cells, and, as such, provides new insight into the cells were treated with ODQ (0–30 mM, as above) and mechanisms by which these cells may circumvent the whole-cell lysates were analysed for MDM2, total p53, normal induction of apoptosis. Moreover, the current and phospho-p53 (Ser15). As shown in Figure 5b, ODQ study suggests that p53 may be intimately involved in upregulated p53, and p53 was phosphorylated on Ser15. the apoptosis resulting from decreased sGC activity/ Furthermore, MDM2 was also upregulated by ODQ, cGMP availability. which was expected given its p53-responsive nature. While ODQ-induced apoptosis was inhibited by both Taken together, these results suggest that inhibition of ANF, an activator of the particulate form of guanylyl the sGC/cGMP pathway upregulates p53 and induces cyclase (pGC), and the stable cGMP analog 8-Br- p53-dependent apoptosis via phosphorylation-depen- cGMP, neither agent completely blocked ODQ-induced dent protein stabilization. apoptosis, although both reduced the percentage of apoptotic cells by >50%. It is possible that these agents were not able to fully restore basal cGMP levels, and Discussion thus could not completely restore cGMP-dependent cell survival. Moreover, 8-Br-cGMP is relatively, but not The present study demonstrates that the basal activity of completely, resistant to phosphodiesterase-mediated the sGC/cGMP pathway plays an important role in the degradation. Thus, the effects of this analog may be

Figure 5 sGC/cGMP regulates p53 stability and phosphorylation. (a) A2780s cells were ‘pulsed’ with ODQ (30 mM) or DMSO (1:1000) for 6 h. Media were removed and cells were treated with cycloheximide (10 mg/ml) for different durations as indicated. Whole- cell lysates were analysed for p53 content by Western blotting. Mean p53 contents (fold of 0 h control for each treatment group) of three independent experiments is included beneath the representative Western blot. (b) A2780s cells were treated with ODQ (30 mM)or DMSO (1:1000) for 24 h, and p53, phospho-p53 (Ser15), and MDM2 contents were analysed by Western blotting. Blots shown are representative of three independent replicates.

Oncogene cGMP regulates p53 and apoptosis in ovarian cancer cells M Fraser et al 2209 limited by degradation. Furthermore, ANF receptor et al., 2002), the mechanism by which this occurs is not abundance in ovarian cancer cells has not been known. Moreover, Heinloth et al did not examine if p53 examined, and may limit the effectiveness of this downregulation was involved in cGMP-mediated cell activator. survival. In the current study, we demonstrate that Xiap overexpression attenuated ODQ-induced apop- inactivation of sGC/cGMP signalling by ODQ treat- tosis. Together with the finding that ODQ-induced ment increases p53 stability, up-regulates p53 and p53- apoptosis is associated with caspase-3 cleavage, this dependent apoptosis. Interestingly, ODQ also induced result suggests that ODQ-induced apoptosis may be the phosphorylation of p53 on Ser15, a residue that caspase-mediated. This is consistent with previous data lies adjacent tothe MDM2-binding site. It is required (Flamigni et al., 2001) that ODQ activates caspase-3 and for p53 stabilization and activation following cell stress -9, although an involvement of these caspases in ODQ- and is a determinant of p53-mediated apoptosis (Shieh induced apoptosis was not demonstrated. Our data et al., 1997; Siliciano et al., 1997; Ashcroft et al., 1999; suggest a functional role for these caspases in ODQ- Unger et al., 1999; Persons et al., 2000). Since ODQ also induced apoptosis, and provides evidence that Xiap is a upregulated MDM2, it is possible that depletion of key regulator of apoptosis in human ovarian cancer sGC-derived cGMP results in phosphorylation-depen- cells. dent attenuation of MDM2-mediated p53 degradation. p53 regulates the expression of genes involved in It is alsopossible that sGC-mediated cell survival results DNA repair, cell cycle arrest, and apoptosis. It is from cGMP-dependent inhibition of another kinase upregulated during cell stress via a phosphorylation- targeting p53. Future studies will clarify the mechanism dependent process which prevents p53 degradation by by which the sGC/cGMP pathway enhances p53 its negative regulator, MDM2. Since MDM2 is a p53- stability. responsive gene product, this system functions as a Panahian et al. (1999) demonstrated that overexpres- negative feedback loop, avoiding inappropriate induc- sion of heme oxygenase-1 (HO-1) facilitates neuropro- tion of p53-mediated cell cycle arrest or apoptosis but tection, an effect associated with cGMP upregulation permitting a rapid response to cell stress. Moreover, the and inhibition of p53 nuclear localization. Moreover, MDM2–p53 interaction is promoted by a number of cell HO-1 colocalizes with p53 in the cytoplasm of motor survival mediators (Mayo and Donner, 2001; Zhou neurons below the level of injury and with cGMP in the et al., 2001; Ogawara et al., 2002). p53 alsocontributes nucleus of injured cells (Panahian and Maines, 2001). to apoptosis by a transcription-independent mechanism, However, these results do not directly implicate cGMP likely via translocation to the mitochondria, where it as a regulator of cell survival, or as a regulator of p53. In binds toand inhibits the antiapoptoticBcl-2 family addition, although NO-induced apoptosis is associated members (Mihara et al., 2003; Erster et al., 2004). with an upregulation of p53 and Bax, these responses ODQ upregulated p53 in three ovarian cancer cell were cGMP-independent (Chen et al., 2001). Thus, lines, and this was attenuated by cotreatment with 8-Br- while some correlative evidence exists to suggest a link cGMP, suggesting that maintenance of low basal p53 between cGMP and p53, our report represents the first levels is promoted by tonic sGC activation and cGMP direct evidence demonstrating that the sGC/cGMP production. 8-Br-cGMP had no effect on the basal levels pathway facilitates cell survival by negatively regulating of p53. This is consistent with the hypothesis that basal p53 content/function. Indeed, one previous study p53 levels are already tightly regulated, and cannot be demonstrates that activation of the cGMP pathway lowered further. However, removal of basal cGMP by upregulates p53 and induces apoptosis in rat vascular inhibition of sGC eliminates its tonic inhibitory effect, endothelial cells (Suenobu et al., 1999). Thus, the role of and p53 levels are increased. Intriguingly, ODQ the sGC/cGMP pathway as a cell fate determinant may upregulated p53 in the cisplatin-resistant C13* cell line. be cell-type specific. We previously showed that cisplatin does not markedly The Akt cell survival pathway plays an important role upregulate p53 in these cells (Fraser et al., 2003), in regulating p53 in human ovarian cancer cells (Fraser suggesting that the mechanism of p53 upregulation by et al., 2003). Akt phosphorylates and activates MDM2 ODQ is distinct from that of cisplatin. Moreover, ODQ- (Mayoand Donner,2001; Zhou et al., 2001; Ogawara induced apoptosis was attenuated by the specific p53 et al., 2002) and cGMP promotes Akt phosphorylation inhibitor PFT, by p53 RNAi, and in p53 mutant cells. (Ciani et al., 2002; Falcone et al., 2002; Ha et al., 2003; All of these mechanisms of p53 inhibition attenuated Tejedo et al., 2004). Thus, it is possible that ODQ- ODQ-induced apoptosis by B50%, suggesting that mediated cGMP depletion inactivates Akt, thereby while p53 contributes to ODQ-induced apoptosis, p53- attenuating the inhibitory effect of MDM2 on p53. independent mechanism(s) are likely involved. Since However, we did not observe any effects of ODQ on ODQ also downregulated Xiap, this may represent one phospho- or total Akt contents (data not shown), of these p53-independent mechanisms. This aspect is suggesting that the ODQ-induced apoptosis are not currently under investigation. mediated via inactivation of Akt. It is intriguing that Thus, our study provides the first direct evidence ODQ-induced apoptosis is significantly, although in- that the sGC/cGMP pathway can regulate apoptosis, completely, attenuated by 8-Br-cGMP. While we cannot in part, via regulation of p53 content/function. While completely exclude the possibility that ODQ may have 8-Br-cGMP downregulates p53 following treatment of nonspecific actions which result in apoptosis, the find- macrophages with oxidized LDL (OxLDL) (Heinloth ing that p53 content was returned to basal levels by

Oncogene cGMP regulates p53 and apoptosis in ovarian cancer cells M Fraser et al 2210 8-Br-cGMP suggests that this effect of ODQ, and the Inc. (Beverly, MA, USA). Rabbit polyclonal anti-Xiap anti- resulting p53-mediated apoptosis, is specific to the body was from Trevigen, Inc. (Gaithersberg, MD, USA). reduction in cGMP levels. Mouse monoclonal anti-GAPDH (glyceraldehyde phosphate Endogenous NO depletion upregulates p53 and dehydrogenase) antibody (ab8245) was from Abcam (Cam- sensitizes melanoma cells to cisplatin-induced apoptosis bridge, UK). Prestained SDS–PAGE standards were from Bio- Rad (Hercules, CA, USA). ODQ and 8-Bromo-cGMP were (Tang and Grimm, 2004). Since NO functions primarily, from Calbiochem (La Jolla, CA, USA). Scrambled control although not exclusively (Chen et al., 2001), through the siRNA was from Dharmacon Inc. (Lafayette, CO, USA). sGC activation, reduction in NO/sGC-derived cGMP RiboJuice siRNA transfection reagent was purchased from levels may have been responsible for the p53 upregula- Novagen, Inc. (San Diego, CA, USA). Pifithrin-a hydrobro- tion, although this was not examined. NO promotes mide was purchased from Tocris, Inc. (Ellisville, MO, USA). mammary tumor invasion and nitric oxide synthase Xiap-s and LacZ adenoviruses were synthesized in the (NOS) activity is associated with breast cancer progres- laboratory of Dr Ruth Slack, Adenovirus Core Facility, sion (Orucevic et al., 1999; Jadeski et al., 2000; Jadeski University of Ottawa. ANF was a generous gift from Dr et al., 2003). Thus, activation of NO/cGMP may be Adolfo de Bold, University of Ottawa Heart Institute. important for tumorigenesis and cancer progression. Moreover, the basal activity of sGC mediating the Cell lines and culture antiapoptotic effects in human ovarian cancer cells may All cell lines were maintained at 371C and in an atmosphere of be dependent, at least in part, on the endogenous 5% CO2/95% air. Wild-type p53, cisplatin-sensitive A2780s human ovarian cancer cells, and the mutant p53, cisplatin- production of NO. Although the role of endogenous resistant counterpart cell line A2780cp were maintained in NO is not yet clear, recent data from our laboratory Dulbecco’s modified Eagle’s medium (DMEM)/F12 medium. have shown that human ovarian cancer cells express all OV2008 and C13* human ovarian cancer cells were main- three forms of NOS, which likely results in elevated tained in RPMI 1640 medium. All media were supplemented basal sGC activity (unpublished data). with fetal bovine serum (10%), streptomycin (50 g/ml), It will be of interest to study the in vivo relationship penicillin (50 U/ml), fungizone (0.625 g/ml; Life Technologies, between sGC/cGMP, p53, and ovarian tumorigenesis. Inc., BRL, Carlsbad, CA, USA), and nonessential amino acids Little is known regarding the factors that give rise to (1%), as previously reported (Fraser et al., 2003). Cells were 4 2 ovarian tumors, since most are not discovered until plated at a density of 5  10 cells/cm in six-well plates 18 h Stage III or IV, when it is difficult to decipher the prior to experimental treatments. At the time of treatment, the cells were 85% confluent. molecular origins of the tumor. It will be critical to p examine whether molecular abnormalities of sGC/ cGMP are a common event in ovarian cancer, where Hoechst 33258 staining alterations in other apoptosis-related genes and gene At the end of the culture period, cells attached to the growth surface were removed by trypsin treatment (trypsin (0.05%), products (e.g. PI3K, Akt, Bcl-2, p53) are frequently EDTA (0.53 mM); 371C, 1 min). Attached and floating cells observed. Intriguingly, high urinary cGMP levels were pooled, pelleted by centrifugation, and resuspended in correlate with poor prognosis in ovarian and other phosphate-buffered formalin (10%) containing Hoechst 33258 cancers (Luesley et al., 1986; Luesley et al., 1987; Turner (12.5 ng/ml). Cells were spotted onto slides for microscopy. et al., 1982a, b), although if the cGMP is derived Nuclear staining was observed and photographed using a Zeiss from the tumor and plays a causative role in ova- fluorescence microscope (magnification  400). Cells with rian tumorigenesis and therapeutic response remains typical apoptotic nuclear morphology (nuclear shrinkage, unclear. condensation, and fragmentation) were identified and counted In summary, our findings provide a new concept for as previously reported (Sasaki et al., 2000, 2002; Asselin et al., the regulation of apoptosis in ovarian cancer cells and of 2001; Fraser et al., 2003), using randomly selected fields. A minimum of 200 cells were counted in each treatment group. ovarian tumor growth, showing a potential link between The counterwas ‘blinded’ tosample identity toavoid the antiapoptotic effects of the sGC/cGMP signalling experimental bias. pathway and the regulation of p53, an important regulator of tumorigenesis and chemotherapeutic response. Determination of apoptosis by TUNEL assay Apoptosis also was assessed by the TUNEL method using the In Situ Cell Death Detection Kit, TMR Red assay kit from Materials and methods Roche (Laval, QC, Canada), according to the manufacturer’s instructions. Briefly, cells were plated as above and treated Reagents with ODQ (0, 30 mM). After 24 h of treatment, floating and Hoechst 33258, phenylmethylsulfonyl fluoride (PMSF), so- attached cells were pooled and pelleted by centrifugation dium orthovanadate (Na3VO4), aprotinin, and cycloheximide (5 min; 300 g) and washed 2  in phosphate-buffered saline were purchased from Sigma (St Louis, MO, USA). Mouse (PBS). The cells were fixed in 10% phosphate-buffered monoclonal anti-p53 antibodies, clone 80 and clone DO-1, formalin for 1 h on ice. Fixed cells were washed in PBS and were purchased from Transduction Laboratories, Inc. (San permeabilized using 0.1% Triton X-100 in 0.1% sodium citrate Diego, CA, USA) and Santa Cruz Biotechnologies (Santa for 5 min. Cells were resuspended in 50 ml TUNEL reaction Cruz, CA, USA), respectively. Mouse monoclonal Bax anti- mixture (5 ml enzyme þ 45 ml label solution) and incubated at body (clone 2D2) was from Santa Cruz Biotechnologies. 371C in the dark for 1 h. Hoescht 33258 (1:1000) was added Mouse monoclonal anti-p21 (clone DCS60), rabbit anti- during the last 5 min to allow visualization of all nuclei under caspase-3 (cleaved fragment), mouse anti-phospho-p53 florescent microscopy. Cells were washed 2  in PBS and then (Ser15), and p53 siRNA were from Cell Signaling Technology, plated on microscope slides and visualized by florescence

Oncogene cGMP regulates p53 and apoptosis in ovarian cancer cells M Fraser et al 2211 microscopy. The percentage of cells undergoing apoptosis was p53 half-life determination determined by counting the number TUNEL-positive nuclei After 18 h of plating, culture medium was removed and divided by the total number of nuclei as determined by replaced with serum-free medium containing ODQ (30 mM)or Hoescht 33258 staining. DMSO (1:2000) and then cells were returned tothe incubator for 6 h. The medium was then removed, the cells were washed twotimes with serum-free medium and fresh medium was cGMP assay added containing 10 mg/ml cycloheximide. Cells were harvested cGMP contents were determined using Enzyme Immunoassay at various time points following cycloheximide treatment Kits (Direct Cyclic GMP) from Assay Designs, Inc. (Ann (0, 30, 60, 120, 240 min). Whole-cell lysates were obtained and Arbor, MI, USA). The cells were plated onto 100 mm plates p53 content was analysed by Western blot. Blots were and after 2 days, experiments were performed to determine the differentially exposed such that the signal was in the linear cGMP depletion effects of ODQ. Each plate represented range for both treatment groups (ODQ vs DMSO). approximately 7.5 million cells. The cells were exposed to ODQ at 10, 30, 50, and 100 mM for 20 min, followed by rapid Adenoviral infection removal of media, rapid washing of the cells with PBS and After 18 h of plating (106 cells/60-mm culture dish), cells were addition of 1.5 ml of ice-cold 0.1 N HCl. The cells (with the infected with adenoviral sense Xiap (Xiap-s) or LacZ cDNA at HCl cell-lysis solution) were set on ice for 10 min, followed by various multiplicities of infection (MOI) as indicated in the scraping the cells from the plate and transferring the samples text as previously described (Li et al., 2001). Adenovirus tomicrofugetubes. The cell lysates were centrifuged at 600 g at infection efficiency at MOI of 5, as determined by an X-gal room temperature and the supernatant fractions collected for staining assay against LacZ construct infected cells, was cGMP analysis. Both the samples (200 ml) and the standards >90%. Xiap overexpression was confirmed by Western blot (200 ml, alsoprepared in the same 0.1 N HCl solution) were analysis. acetylated (as described in the protocol of the kits) in order to increase the sensitivity of the assay for cGMP measurement. Twoaliquots(100 ml each) of the samples and standards were RNA interference pipeted into the wells of a 96-well tray (provided in the kits). At 18 h after plating, 6 ml of transfection reagent was added to The addition of the antibody (specific for cGMP) and other 244 ml of DMEM/F12 without serum. The mixture was reagents were added as described in the protocol of the kits. To vortexed and incubated for 5 min at room temperature. increase the accuracy of the measurements of cGMP in Following incubation, 7.5 mlof10mM stock siRNA construct samples with low cGMP content (i.e. ODQ-treated cells), we was added. The mixture was incubated at room temperature added two additional low-concentration standards (4 and for a further 15 min. During this period, the culture media were 2 fmoles/well). The number of cells in the 100 mm dish was removed from the cells and the cells were washed once with determined by counting cells with a hemocytometer. cGMP PBS. The siRNA mixture was added in single drops to each contents are reported as fmol/million cells. well to ensure maximal coverage, and an additional 1250 mlof complete (10% FBS) media were added. The cells were returned to the incubator and the media were removed 6 h Protein extraction and Western blot analysis later and replaced with fresh, complete media for the duration Cells were pelleted and lysed in ice-cold lysis buffer (pH 7.4) of the culture (24 h). Downregulation was confirmed by Western blot analysis. containing 50 mM HEPES, 150 mM NaCl, 1.5 mM MgCl2, 1mM EGTA, 100 mM NaF, 10 mM NaPPi, 10% glycerol, and 1% Triton X-100. Protease inhibitors PMSF (1 mM) and Statistical analysis 7 aprotonin (10 g/l), as well as 1 mM Na3VO4 were added tothe Results are expressed as the mean s.e.m. of at least three lysis buffer freshly. Cell lysates were sonicated briefly (5 s/ independent experiments. Statistical analysis was carried out cycle, 3 cycles; 01C). The sonicates were incubated on ice for by one- or two-way ANOVA or by Student’s t-test (where 1 h and pelleted by centrifugation (15 000 g; 20 min). The appropriate) using PRISM software (Version 3.0; GraphPad, supernatant was taken as whole-cell lysate and stored at San Diego, CA, USA). Differences between multiple experi- À201C for subsequent analyses. Protein concentration was mental groups were determined by the Bonferroni post hoc determined using Bio-Rad DC protein assay kit. Equal test. Statistical significance was inferred at **Po0.05. amounts of proteins (30–70 mg) were loaded and resolved by 10% SDS–PAGE and electro-transferred (30 V, 16 h) onto nitrocellulose membranes (Bio-Rad). Membranes were Abbreviations blocked (room temperature, 1 h) with 5% Blotto (Tris-HCl (10 mM; pH 8.0), NaCl (150 mM), Tween 20 (0.05%, v/v; TBS- sGC, soluble guanylyl cyclase; pGC, particulate guanylyl Tween 20) containing skim milk (5%; w/v)), then incubated cyclase; cGMP, cyclic guanosine monophosphate; PKG, overnight with primary antibodies (p53 (1:1000, Clones 80 and protein kinase G; ODQ, 1H-[1,2,4]oxadiazolo[43-a]quinoxa- DO-1), p21 (1:1000), Xiap (1:2000), cleaved caspase-3 (1:1000), lin-1-one; IAP, inhibitor of apoptosis protein; GAPDH, or GAPDH (1:30,000)), and subsequently with the appropriate glyceraldehyde phosphate dehydrogenase; BNP, brain na- horseradish peroxidase (HRP)-conjugated secondary antibody triuretic peptide; ANF, atrial natriuretic factor; SDS–PAGE, (1:2000 in 5% Blotto; room temperature, 1 h; 1:20 000 for sodium dodecyl sulfate–polyacrylamide gel electrophoresis; GAPDH). Peroxidase activity was visualized with an enhanced PMSF, phenylmethylsulfonyl fluoride; RNAi, RNA interfer- chemiluminescence (ECL) kit (Amersham Pharmacia Biotech, ence; siRNA, small inhibitory RNA; DMEM, Dulbecco’s Arlington Heights, IL, USA) after three washes (15 min/wash) modified Eagle’s medium; ECL, enhanced chemiluminescence; with TBS-Tween 20. Signal intensity was determined densito- PFT, pifithrin-a hydrobromide; PBS, phosphate-buffered metrically using Scion Image software, version 4.02 from Scion saline; Xiap, X-linked inhibitor of apoptosis protein; Xiap-s, Corporation (Frederick, MD, USA). Even loading between adenoviral Xiap sense cDNA; ROS, reactive oxygen species; lanes was determined using Ponceau-S stain and by densito- MDM2, murine double minute-2; NO, nitric oxide; CHX, metry of the anti-GAPDH blots. cycloheximide.

Oncogene cGMP regulates p53 and apoptosis in ovarian cancer cells M Fraser et al 2212 Acknowledgements Council of Hong Kong (Grant # CUHK4169/02M) and the Graduate School Bursary Sub-Committee of The Chinese This work was supported in part by grants awarded to University of Hong Kong for the grant for Overseas Academic Benjamin K Tsang by the Canadian Institutes of Health Activities, awarded toSiu Lan Chan. Michael Fraser is Research (MOP-15691) and the National Cancer Institute of the recipient of a Canada Graduate Scholarship Doctoral Canada (with funds from the Canadian Cancer Society, Grant Research Award from the Canadian Institutes of Health 013335) and toRonaldR Fiscus by the Research Grants Research.

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