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Supplementary Fig. S1 from Chemosensitizing effects of sphingosine kinase-1 inhibition in prostate cancer cell and animal models

Supplementary Fig. S2 from Chemosensitizing effects of sphingosine kinase-1 inhibition in prostate cancer cell and animal models

We have previously reported that, in prostate cancer, inhibition of the oncogenic sphingosine kinase-1/sphingosine 1-phosphate (SphK1/S1P) pathway is a key element in chemotherapy-induced apoptosis. Here, we show that selective pharmacologic inhibition of SphK1 triggers apoptosis in LNCaP and PC-3 prostate cancer cells, an effect that is reversed by SphK1 enforced expression. More importantly, we show for the first time that the up-regulation of the SphK1/S1P pathway plays a crucial role in the resistance of prostate cancer cells to chemotherapy. Importantly, pharmacologic SphK1 inhibition with the B-5354c compound sensitizes LNCaP and PC-3 cells to docetaxel and camptothecin, respectively. In vivo, camptothecin and B-5354c alone display a limited effect on tumor growth in PC-3 cells, whereas in combination there is a synergy of effect on tumor size with a significant increase in the ceramide to S1P sphingolipid ratio. To conclude, our study highlights the notion that drugs specifically designed to inhibit SphK1 could provide a means of enhancing the effects of conventional treatment through the prosurvival antiapoptotic SphK1/S1P pathway. [Mol Cancer Ther 2008;7(7):1836–45]

International audience; We examined the role of sphingosine kinase-1 (SphK1), a critical regulator of the ceramide/sphingosine 1-phosphate (S1P) biostat, in the regulation of death and survival of SH-SY5Y neuroblastoma cells in response to amyloid beta (Abeta) peptide (25-35). Upon incubation with Abeta, SH-SY5Y cells displayed a marked down-regulation of SphK1 activity coupled with an increase in the ceramide/S1P ratio followed by cell death. This mechanism was redox-sensitive; N-acetylcysteine totally abrogated the down-regulation of SphK1 activity and strongly inhibited Abeta-induced cell death. SphK1 overexpression impaired the cytotoxicity of Abeta, whereas SphK1 silencing by RNA interference mimicked Abeta-induced cell death, thereby establishing a critical role for SphK1. We further demonstrated that SphK1 could mediate the well established cytoprotective action of insulin-like growth factor (IGF-I) against Abeta toxicity. A dominant-negative form of SphK1 or its pharmacological inhibition not only abrogated IGF-I-triggered stimulation of SphK1 but also hampered IGF-I protective effect. Similarly to IGF-I, the neuroprotective action of TGF-beta1 was also dependent on SphK1 activity; activation of SphK1 as well as cell survival were impeded by a dominant-negative form of SphK1. Taken together, these results provide the first illustration of SphK1 role as a critical regulator of death and survival of Abeta-treated cells.

Systemic chemotherapy was considered of modest efficacy in prostate cancer until the recent introduction of taxanes. We took advantage of the known differential effect of camptothecin and docetaxel on human PC-3 and LNCaP prostate cancer cells to determine their effect on sphingosine kinase-1 (SphK1) activity and subsequent ceramide/sphingosine 1-phosphate (S1P) balance in relation with cell survival. In vitro, docetaxel and camptothecin induced strong inhibition of SphK1 and elevation of the ceramide/S1P ratio only in cell lines sensitive to these drugs. SphK1 overexpression in both cell lines impaired the efficacy of chemotherapy by decreasing the ceramide/S1P ratio. Alternatively, silencing SphK1 by RNA interference or pharmacologic inhibition induced apoptosis coupled with ceramide elevation and loss of S1P. The differential effect of both chemotherapeutics was confirmed in an orthotopic PC-3/green fluorescent protein model established in nude mice. Docetaxel induced a stronger SphK1 inhibition and ceramide/S1P ratio elevation than camptothecin. This was accompanied by a smaller tumor volume and the reduced occurrence and number of metastases. SphK1-overexpressing PC-3 cells implanted in animals developed remarkably larger tumors and resistance to docetaxel treatment. These results provide the first in vivo demonstration of SphK1 as a sensor of chemotherapy. (Cancer Res 2005; 65(24): 11667-75)

We examined the involvement of sphingosine kinase-1, a critical regulator of the sphingolipid balance, in susceptibility to antineoplastic agents of either sensitive or multidrug-resistant acute myeloid leukemia cells. Contrary to parental HL-60 cells, doxorubicin and etoposide failed to trigger apoptosis in chemoresistant HL-60/Doxo and HL-60NP16 cells overexpressing MRP1 and MDR1, respectively. Chemosensitive HL-60 cells displayed sphingosine kinase-1 inhibition coupled with ceramide generation. In contrast, chemoresistant HL-60/ Doxo and HL-60/VP16 had sustained sphingosine kinase-1 activity and did not produce ceramide during treatment. Enforced expression of sphingosine kinase-1 in chemosensitive HL-60 cells resulted in marked inhibition of apoptosis that was mediated by blockade of mitochondrial cytochrome c efflux hence suggesting a control of apoptosis at the pre-mitochondrial level. Incubation with cell-permeable ceramide of chemoresistant cells led to a sphingosine kinase-1 inhibition and apoptosis both prevented by sphingosine kinase-1 over-expression. Furthermore, F-12509a, a new sphingosine kinase inhibitor, led to ceramide accumulation, decrease in sphingosine 1-phosphate content and caused apoptosis equally in chemosensitive and chemoresistant cell lines that is inhibited by adding sphingosine 1-phosphate or overexpressing sphingosine kinase-1. F-12509a induced classical apoptosis hallmarks namely nuclear fragmentation, caspase-3 cleavage as well as downregulation of antiapoptotic XIAP, and release of cytochrome c and SMAC/Diablo.

The signaling cascade resulting in the death of several types of cells treated with ouabain or other cardiotonic steroids (CTS) remains poorly understood. Recently, we observed that ouabain kills epithelial and endothelial cells via its interaction with Na(+), K(+) -ATPase, but independently of inhibition of Na(+), K(+) -ATPase-mediated ion fluxes and inversion of the [Na(+)](i)/[K(+)](i) ratio. Here, we report that the death of ouabain-treated epithelial cells from the Madin-Darby canine kidney (C7-MDCK) and endothelial cells from porcine aortae is suppressed by acidification of medium from pH 7.4 to 7.0, i.e. under conditions when pH(i) was decreased from approximately 7.2 to 6.9. The rescue of ouabain-treated C7-MDCK cells was also detected under selective intracellular acidification caused by inhibition of Na(+)/H(+) exchanger. In these cells, neither Na(+), K(+) pump activity nor [(3)H]-ouabain binding was significantly affected by modest acidification. The death of ouabain-treated cells was independent of inhibition of RNA and protein synthesis with actinomycin D and cycloheximide. In contrast, both compounds sharply attenuated the protective action of acidified medium. Thus, our results show that very modest intracellular acidification is sufficient to inhibit the Na(+) (i)/K(+) (i)-independent death signal triggered in epithelial and endothelial cells by CTS. They also suggest that the protective action of acidification is mediated by de novo expression of genes involved in inhibition of the cell death machinery.

Recent studies have demonstrated the tissue-specific effect of Na+/K+ pump inhibition by ouabain and other cardiac glycosides on cell viability. The vascular endothelium is an initial target of cardiac glycosides employed for the management of congestive heart failure as well as circulating endogenous ouabain-like substances (EOLS), the production of which is augmented in volume-expanded hypertension. This study examined the role of the Na+/K+ pump in the survival of cultured porcine aortic endothelial cells (PAEC). Complete Na+/K+ pump inhibition with ouabain led to PAEC death, indicated by cell detachment and decreased staining with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Based on cell swelling and resistance to benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (z-VAD.fmk) a pan-caspase inhibitor, this type of cell death was classified as necrosis. In contrast to ouabain, Na+/K+ pump inhibition in K+-free medium did not affect PAEC viability and sharply attenuated apoptosis triggered by 3H decay-induced DNA damage. Necrosis evoked by ouabain was preserved after dissipation of the transmembrane gradient of K+ and Na+, whereas dissipation of the Na+ gradient abolished the antiapoptotic action of K+-free medium. Comparative analysis of these results and modulation of intracellular Na+ and K+ content by the above-listed stimuli showed that interaction of ouabain with Na+/K+-ATPase triggered necrosis independently of inhibition of Na+/K+ pump-mediated ion fluxes and inversion of the [Na+]i/[K+]i ratio, whereas protection against apoptosis under Na+/K+ pump inhibition in K+-depleted medium was mediated by [Na+]i elevation. The role of Na+/K+ pump-mediated regulation of endothelial cell survival and vascular remodelling seen in hypertension should be investigated further in context of EOLS and chronic treatment with digitalis.

Shrinkage is the earliest hallmark of cells undergoing apoptosis. This study examines the role of this phenomenon in the onset of vascular smooth muscle cell (VSMC) apoptosis triggered by growth factor withdrawal. In hyperosmotic media, VSMC showed the same amplitude of shrinkage but were more resistant to apoptosis than endothelial, epithelial and immune system cells. As with growth factor withdrawal, apoptosis in hyperosmotically-shrunken VSMC was sharply potentiated by transfection with E1A-adenoviral protein and was suppressed by activation of cAMP signaling as well as by the pan-caspase inhibitor z-VAD.fmk. Both cell shrinkage and apoptosis in VSMC-E1A treated with hyperosmotic medium were potentiated under sustained Na+, K+ pump inhibition with ouabain that was in contrast to inhibition of apoptosis documented in ouabain-treated, serum-deprived cells. After 1-hr incubation in serum-deprived medium, VSMC-E1A volume declined by approximately 15%. Transfer from hypotonic to control medium decreased VSMC-E1A volume by approximately 25% without any induction of apoptosis. Neither swelling in hyposmotic medium nor dissipation of the transmembrane gradient of K+ and major organic osmolytes protected serum-deprived VSMC-E1A from apoptosis. Thus, our results show that similarly to immune system, endothelial and epithelial cells, extensive VSMC shrinkage in hyperosmotic medium leads to the development of apoptosis. In contrast to hyperosmotic medium, the modest cell volume decrease occurring in serum-deprived VSMC does not contribute to triggering of the apoptotic machinery.

[(3)H]-thymidine is commonly used to analyze the accumulation of [(3)H]-labeled chromatin fragments in cells undergoing apoptosis. This study shows that [(3)H]-thymidine incorporation within DNA is sufficient per se to inhibit growth and to induce apoptosis in canine kidney epithelial cells and porcine aorta endothelial cells. Despite high-level [(3)H]-thymidine-DNA labeling, rat vascular smooth muscle cells (VSMC) showed only modest inhibition of growth and induction of apoptosis compared to other cell types. Similarly to serum deprivation, apoptosis triggered by [(3)H]-thymidine labeling was sharply potentiated by VSMC transfection with a functional analogue of c-myc, E1A-adenoviral protein (VSMC-E1A), and was suppressed by stimulation of cAMP signaling with forskolin as well as by and Na/K pump inhibition with ouabain. Both apoptosis induction and growth suppression seen in [(3)H]-thymidine-treated VSMC-E1A were reduced by the pan-caspase inhibitor z-VAD.fmk. Thus, our results show that the differential efficiency of the apoptotic machinery determines cell type-specific attenuation of growth in cells with [(3)H]-thymidine-labeled DNA. They also demonstrate that [(3)H]-thymidine-treated and serum-deprived VSMC employ common intermediates of the apoptotic machinery, including steps that are potentiated by E1A-adenoviral protein and inhibited by activation of cAMP signaling as well as by inversion of the intracellular [Na(+)](i)/[K(+)](i) ratio.

Treatment with ouabain led to massive death of principal cells from collecting ducts (C7-MDCK), indicated by cell swelling, loss of mitochondrial function, an irregular pattern of DNA degradation, and insensitivity to pan-caspase inhibitor. Equimolar substitution of extracellular Na(+) by K(+) or choline(+) sharply attenuated the effect of ouabain on intracellular Na(+) and K(+) content but did not protect the cells from death in the presence of ouabain. In contrast to ouabain, inhibition of the Na(+)/K(+) pump in K(+)-free medium increased Na(+)(i) content but did not affect cell survival. In control and K(+)-free medium, ouabain triggered half-maximal cell death at concentrations of approximately 0.5 and 0.05 microM, respectively, which was consistent with elevation of Na(+)/K(+) pump sensitivity to ouabain in K(+)-depleted medium. Our results show for the first time that the death of ouabain-treated renal epithelial cells is independent of the inhibition of Na(+)/K(+) pump-mediated ion fluxes and the [Na(+)](i)]/[K(+)](i) ratio.

In this study, we examined the effect of Na(+)-K(+) pump inhibition on the expression of early response genes in vascular smooth muscle cells (VSMC) as possible intermediates of the massive RNA synthesis and protection against apoptosis seen in ouabain-treated VSMC in our previous experiments. Incubation of VSMC with ouabain resulted in rapid induction of c-Fos protein expression with an approximately sixfold elevation after 2 h of incubation. c-Jun expression was increased by approximately fourfold after 12 h, whereas expression of activating transcription factor 2, cAMP/Ca(2+) response element binding protein (CREB)-1 and c-Myc was not altered. Markedly augmented c-Fos expression was also observed under Na(+)-K(+) pump inhibition in potassium-depleted medium. Na(+)-K(+) pump inhibition triggered c-Fos expression via elevation of the [Na(+)](i)/[K(+)](i) ratio. This conclusion follows from experiments showing the lack of effect of ouabain on c-Fos expression in high-potassium-low-sodium medium and from the comparison of dose responses of Na(+)-K(+) pump activity, [Na(+)](i) and [K(+)](i) content and c-Fos expression to ouabain. A fourfold increment of c-Fos mRNA was revealed 30 min following addition of ouabain to the incubation medium. At this time point, treatment with ouabain resulted in an approximately fourfold elevation of [Na(+)](i) but did not affect [K(+)](i). Augmented c-Fos expression was also observed under VSMC depolarization in high-potassium medium. Increments in both c-Fos expression and (45)Ca uptake in depolarized VSMC were abolished under inhibition of L-type Ca(2+) channels with 0.1 microM nicardipine. Ouabain did not affect the free [Ca(2+)](i) or the content of exchangeable [Ca(2+)](i). Ouabain-induced c-Fos expression was also insensitive to the presence of nicardipine and [Ca(2+)](o), as well as chelators of [Ca(2+)](o) (EGTA) and [Ca(2+)](i) (BAPTA). The effect of ouabain and serum on c-Fos expression was additive. In contrast to serum, however, ouabain failed to activate the Elk-1, serum response factor, CREB and activator protein-1 transcription factors identified within the c-Fos promoter. These results suggest that Na(+)-K(+) pump inhibition triggers c-Fos expression via [Na(+)](i)-sensitive [Ca(2+)](i)-independent transcription factor(s) distinct from factors interacting with known response elements of this gene promoter.

Purpose: Sphingosine kinase-1 (SphK1) was shown in preclinical models and non-genitourinary cancers to be instrumental in cancer progression, adaptation to hypoxia and in tumour angiogenesis. No data were available in human prostate cancer. The present study was designed to assess SphK1 expression and activity in radical prostatectomy specimens and to research correlations with clinical features.Materials and methods: Transverse section of fresh tissue was obtained from 30 consecutive patients undergoing laparoscopic prostatectomy. SphK1 enzymatic activities of tumour and normal counterpart were determined. Relationships with PSA, Gleason sum, pathological stage, resection margin status and treatment failure were researched. SphK1 pattern ofexpression was then assessed on tissue microarray. Results: A significant 2-fold increase in SphK1 enzymatic activity(11.1 ± 8.4 versus 5.9 ± 3.2(P < 0.04)) was observed in cancer. The upper quartile of SphK1 activity was associated with higher PSA (16.7 versus 6.4 ng/ml, P = 0.04), higher tumor volumes (20.7 versus 9.8,P = 0.002), higher rates of positive margins (85.7% versus 28.6%, P = 0.01) and surgical failure(71.4% versus 9.5%, P = 0.003) than the lower three quartiles. Odds ratios (OR) for treatment failure showed a strong relationship with SphK1 activity (OR: 23.7, P = 0.001), positive resection margins (OR: 15.0, P = 0.007) and Gleason sum (P4 + 3, OR: 8.0, P = 0.003). Tissue microarrays showed discrete epithelial expression that varied with Gleason sum with significant relationship between SphK1 expression and higher Gleason sum.Conclusion: In complement to preclinical literature, the demonstrated relationships between SphK1-increased activity in cancer and relevant clinical features confirm a central role for SphK1 in prostate cancer that herald promising avenues in risk-assessment and treatment. Refereed/Peer-reviewed

Defeating pancreatic cancer resistance to the chemotherapeutic drug gemcitabine remains a challenge to treat this deadly cancer. Targeting the sphingolipid metabolism for improving tumor chemosensitivity has recently emerged as a promising strategy. The fine balance between intracellular levels of the prosurvival sphingosine-1-phosphate (S1P) and the proapoptotic ceramide sphingolipids determines cell fate. Among enzymes that control this metabolism, sphingosine kinase-1 (SphK1), a tumor-associated protein overexpressed in many cancers, favors survival through S1P production, and inhibitors of SphK1 are used in ongoing clinical trials to sensitize epithelial ovarian and prostate cancer cells to various chemotherapeutic drugs. We here report that the cellular ceramide/S1P ratio is a critical biosensor for predicting pancreatic cancer cell sensitivity to gemcitabine. A low level of the ceramide/S1P ratio, associated with a high SphK1 activity, correlates with a robust intrinsic pancreatic cancer cell chemoresistance toward gemcitabine. Strikingly, increasing the ceramide/S1P ratio, by using pharmacologic (SphK1 inhibitor or ceramide analogue) or small interfering RNA-based approaches to up-regulate intracellular ceramide levels or reduce SphK1 activity, sensitized pancreatic cancer cells to gemcitabine. Conversely, decreasing the ceramide/S1P ratio, by up-regulating SphK1 activity, promoted gemcitabine resistance in these cells. Development of novel pharmacologic strategies targeting the sphingolipid metabolism might therefore represent an interesting promising approach, when combined with gemcitabine, to defeat pancreatic cancer chemoresistance to this drug.[Mol Cancer Ther 2009;8(4):809–20]

The mitochondrial enzyme monoamine oxidase (MAO), its isoform MAO-A, plays a major role in reactive oxygen species–dependent cardiomyocyte apoptosis and postischemic cardiac damage. In the current study, we investigated whether sphingolipid metabolism can account for mediating MAO-A– and reactive oxygen species–dependent cardiomyocyte apoptosis. In H9c2 cardiomyoblasts, MAO-A–dependent reactive oxygen species generation led to mitochondria-mediated apoptosis, along with sphingosine kinase-1 (SphK1) inhibition. These phenomena were associated with generation of proapoptotic ceramide and decrease in prosurvival sphingosine 1-phosphate. These events were mimicked by inhibition of SphK1 with either pharmacological inhibitor or small interfering RNA, as well as by extracellular addition of C 2 -ceramide or H 2 O 2 . In contrast, enforced expression of SphK1 protected H9c2 cells from serotonin- or H 2 O 2 -induced apoptosis. Analysis of cardiac tissues from wild-type mice subjected to ischemia/reperfusion revealed significant upregulation of ceramide and inhibition of SphK1. It is noteworthy that SphK1 inhibition, ceramide accumulation, and concomitantly infarct size and cardiomyocyte apoptosis were significantly decreased in MAO-A–deficient animals. In conclusion, we show for the first time that the upregulation of ceramide/sphingosine 1-phosphate ratio is a critical event in MAO-A–mediated cardiac cell apoptosis. In addition, we provide the first evidence linking generation of reactive oxygen species with SphK1 inhibition. Finally, we propose sphingolipid metabolites as key mediators of postischemic/reperfusion cardiac injury.

The erythroleukemia developed by spi-1/PU.1-transgenic mice is a model of multistage oncogenic process. Isolation of tumor cells representing discrete stages of leukemic progression enables the dissection of some of the critical events required for malignant transformation. To elucidate the molecular mechanisms of multistage leukemogenesis, we developed a microarray transcriptome analysis of nontumorigenic (HS1) and tumorigenic (HS2) proerythroblasts from spi-1-transgenic mice. The data show that transcriptional up-regulation of the sphingosine kinase gene (SPHK1) is a recurrent event associated with the tumorigenic phenotype of these transgenic proerythroblasts. SPHK1 is an enzyme of the metabolism of sphingolipids, which are essential in several biologic processes, including cell proliferation and apoptosis. HS1 erythroleukemic cells engineered to overexpress the SPHK1 protein exhibited growth proliferative advantage, increased clonogenicity, and resistance to apoptosis in reduced serum level by a mechanism involving activation of the extracellular signal-related kinases 1/2 (ERK1/2) and phosphatidylinositol 3-kinase (PI3K)/AKT pathways. In addition, SPHK1-overexpressing HS1 cells acquired tumorigenicity when engrafted in vivo. Finally, enforced expression of a dominant-negative mutant of SPHK1 in HS2 tumorigenic cells or treatment with a pharmacologic inhibitor reduced both cell growth and apoptosis resistance. Altogether, these data suggest that overexpression of the sphingosine kinase may represent an oncogenic event during the multistep progression of an erythroleukemia. (Blood. 2005;106:1808-1816)

Purinergic inhibition of Na-K-Cl cotransport has been noted in various renal epithelial cells derived from the collecting tubule, including Madin-Darby canine kidney (MDCK) cells. In recent studies, we have observed purinergic inhibition of Na-K-Cl cotransport in C11-MDCK subclones (α-intercalated-like cells). Interestingly, Na-K-Cl cotransport activity was also detected in C7-MDCK subclones (principal-like cells) but was not affected by ATP. In this investigation, we have transfected the human Na-K-Cl cotransporter (huNKCC1) in both C11 and C7 cells to determine whether these differences in NKCC regulation by ATP were due to cell-specific purinoceptor signaling pathways or to cell-specific isoforms/splice variants of the transporter. In both cell lines, we found that endogenous as well as huNKCC1-derived cotransport activity was restricted to the basolateral side. In addition, we were able to show that extracellular application of 100 μM ATP or 100 μM UTP abolished NKCC activity in both mock- and huNKCC1-transfected C11 cells but not in mock- and huNKCC1-transfected C7 cells; in C11 cells, intriguingly, this inhibition was not affected by inhibitors of RNA and protein synthesis and occurred even though expression levels of UTP-sensitive P2Y2-, P2Y4-, and P2Y6-purinoceptors were not different from those observed in C7 cells. These results suggest that C11 cells express an undetermined type of UTP-sensitive P2-purinoceptors or a unique P2Y-purinoceptor-triggered signaling cascade that leads to inhibition of NKCC1.

Background The prognosis of gastric cancer with peritoneal metastasis is extremely poor. Neither systemic chemotherapy nor surgery alone prolongs survival of patients significantly. Methods Patients diagnosed with advanced gastric cancer underwent staging laparoscopy and received chemotherapy when peritoneal dissemination and/or cancer cells on peritoneal cytology were confirmed. The chemotherapy regimen consisted of S-1, weekly intravenous and intraperitoneal paclitaxel, which was verified in our phase II trial (Ann Oncol 2009). S-1 was administered at 80 mg/m2/day for 14 consecutive days, followed by 7 days rest. Paclitaxel was administered intravenously at 50 mg/m2 and intraperitoneally at 20 mg/m2 on days 1 and 8. Clinical response of chemotherapy was assessed by computed tomography, gastroendoscopy, peritoneal cytology and second-look laparoscopy. Radical gastrectomy was carried out when macroscopic curative resection was made achievable by chemotherapy. Chemotherapy was restarted after operation as soon as possible. Overall survival, relapse free survival, morbidity and mortality of gastrectomy were evaluated. Results Out of 100 patients with peritoneal metastasis who received chemotherapy, 60 patients underwent gastrectomy after response to chemotherapy, including 54 with macroscopic metastasis and 6 with positive peritoneal cytology only. A median of three courses were administered preoperatively (range 1–16). Total or distal gastrectomy with lymphnode dissection was carried out in 54 or 6 patients, respectively. The median survival time was 34.5 months. The median relapse-free survival was 16.7 months. The first site of relapse was the peritoneum in 24 patients and the other organ site in 17 patients. Postoperative complications included anastomotic leakage and pancreatic fistula in two patients each, which were healed conservatively. There were no treatment-related deaths. Conclusions Gastrectomy combined with S-1, intravenous and intraperitoneal paclitaxel is safe and active for gastric cancer patients with peritoneal metastasis.

Objective: Endothelial cell damage contributes to vascular remodeling seen in hypertension and other cardiovascular diseases. Previously, we reported that elevation of the [Na+]i/[K+]i ratio protects vascular smooth muscle cells against apoptosis. This study examines the role of intracellular monovalent cations in apoptosis of porcine aorta endothelial cells (PAEC). Design and Methods: Apoptosis in cultured freshly-isolated PAEC was estimated by MTT assay and caspase-3 activity. [14C]-urea available space and atomic absorption spectrophotometry were employed to measure cell volume and intracellular Na and K content, respectively. Results: 24-hr treatment of PAEC with 5 uCi/ml of [3H]-thymidine led to 2-fold attenuation of MTT staining and 10-fold elevation of caspase-3 activity. Apoptosis in [3H]-thymidine-treated cells was abolished by nonradioactive thymidine, the pan-caspase inhibitor z-VAD.fmk and by Na,KATPase inhibition in K+ free medium. Incubation of PAEC in K+ free medium led to 10-fold elevation of Na + i and to 8-fold decrease of K + i. Inhibition of the Na,K-ATPase in K + free Na + depleted medium resulted in the same loss of K + i, but led to modest (2-fold) elevation of Na + i and did not protect PAEC against apoptosis. Conclusions: Extensive DNA labeling with [3H]-thymidine is sufficient to induce apoptosis in PAEC. Elevation of [Na+]i protects PAEC from apoptosis triggered by [3H]-decay-induced DNA damage at a step upstream of caspase-3. These data should be considered under analysis of the involvement of endogenous Na+,K+ ATPase inhibitors and other potent Na + i raising compounds in endothelial cell survival and vascular remodeling seen in hypertension.