Upregulation of ABC transporters contributes to chemoresistance of sphingosine 1-phosphate lyase-deficient fibroblasts
2014; Elsevier BV; Volume: 56; Issue: 1 Linguagem: Inglês
10.1194/jlr.m052761
ISSN1539-7262
AutoresKatja Ihlefeld, Hans Vienken, Ralf Frederik Claas, Kira Blankenbach, Agnes Rudowski, Michael ter Braak, Alexander Koch, Paul P. Van Veldhoven, Josef Pfeilschifter, Dagmar Meyer zu Heringdorf,
Tópico(s)Drug Transport and Resistance Mechanisms
ResumoSphingosine 1-phosphate (S1P) is an extra- and intracellular mediator that regulates cell growth, survival, migration, and adhesion in many cell types. S1P lyase is the enzyme that irreversibly cleaves S1P and thereby constitutes the ultimate step in sphingolipid catabolism. It has been reported previously that embryonic fibroblasts from S1P lyase-deficient mice (Sgpl1−/−-MEFs) are resistant to chemotherapy-induced apoptosis through upregulation of B cell lymphoma 2 (Bcl-2) and Bcl-2-like 1 (Bcl-xL). Here, we demonstrate that the transporter proteins Abcc1/MRP1, Abcb1/MDR1, Abca1, and spinster-2 are upregulated in Sgpl1−/−-MEFs. Furthermore, the cells efficiently sequestered the substrates of Abcc1 and Abcb1, fluo-4 and doxorubicin, in subcellular compartments. In line with this, Abcb1 was localized mainly at intracellular vesicular structures. After 16 h of incubation, wild-type MEFs had small apoptotic nuclei containing doxorubicin, whereas the nuclei of Sgpl1−/−-MEFs appeared unchanged and free of doxorubicin. A combined treatment with the inhibitors of Abcb1 and Abcc1, zosuquidar and MK571, respectively, reversed the compartmentalization of doxorubicin and rendered the cells sensitive to doxorubicin-induced apoptosis. It is concluded that upregulation of multidrug resistance transporters contributes to the chemoresistance of S1P lyase-deficient MEFs. Sphingosine 1-phosphate (S1P) is an extra- and intracellular mediator that regulates cell growth, survival, migration, and adhesion in many cell types. S1P lyase is the enzyme that irreversibly cleaves S1P and thereby constitutes the ultimate step in sphingolipid catabolism. It has been reported previously that embryonic fibroblasts from S1P lyase-deficient mice (Sgpl1−/−-MEFs) are resistant to chemotherapy-induced apoptosis through upregulation of B cell lymphoma 2 (Bcl-2) and Bcl-2-like 1 (Bcl-xL). Here, we demonstrate that the transporter proteins Abcc1/MRP1, Abcb1/MDR1, Abca1, and spinster-2 are upregulated in Sgpl1−/−-MEFs. Furthermore, the cells efficiently sequestered the substrates of Abcc1 and Abcb1, fluo-4 and doxorubicin, in subcellular compartments. In line with this, Abcb1 was localized mainly at intracellular vesicular structures. After 16 h of incubation, wild-type MEFs had small apoptotic nuclei containing doxorubicin, whereas the nuclei of Sgpl1−/−-MEFs appeared unchanged and free of doxorubicin. A combined treatment with the inhibitors of Abcb1 and Abcc1, zosuquidar and MK571, respectively, reversed the compartmentalization of doxorubicin and rendered the cells sensitive to doxorubicin-induced apoptosis. It is concluded that upregulation of multidrug resistance transporters contributes to the chemoresistance of S1P lyase-deficient MEFs. Intrinsic or acquired chemoresistance is one of the major problems of modern tumor therapy. Chemoresistance of tumor cells can be caused by several mechanisms such as induction of drug metabolism, upregulation of multidrug transporters, modification of drug targets, cell cycle arrest, regulation of DNA replication and repair, and modulation of apoptosis (1Fodale V. Pierobon M. Liotta L. Petricoin E. Mechanism of cell adaptation: when and how do cancer cells develop chemoresistance?.Cancer J. 2011; 17: 89-95Crossref PubMed Scopus (150) Google Scholar). About two decades of research now have demonstrated that sphingolipids play a role in the regulation of cell survival, apoptosis, and chemo- or radioresistance, with general consensus identifying ceramide as a proapoptotic and sphingosine 1-phosphate (S1P) as an antiapoptotic mediator (2Young M.M. Kester M. Wang H-G. Sphingolipids: regulators of crosstalk between apoptosis and autophagy.J. Lipid Res. 2013; 54: 5-19Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar, 3Truman J-P. García-Barros M. Obeid L.M. Hannun Y.A. Evolving concepts in cancer therapy through targeting sphingolipid metabolism.Biochim. Biophys. Acta. 2014; 1841: 1174-1188Crossref PubMed Scopus (92) Google Scholar). According to the sphingolipid biostat model, the equilibrium between S1P and ceramide regulates cell fate decisions (4Cuvillier O. Pirianov G. Kleuser B. Vanek P.G. Coso O.A. Gutkind S. Spiegel S. Suppression of ceramide-mediated programmed cell death by sphingosine 1-phosphate.Nature. 1996; 381: 800-803Crossref PubMed Scopus (1347) Google Scholar). Ceramide and S1P are metabolically interconverted by ceramidases and sphingosine kinases (SphKs), phosphatases and ceramide synthases, respectively (5Hannun Y.A. Obeid L.M. Principles of bioactive lipid signalling: lessons from sphingolipids.Nat. Rev. Mol. Cell Biol. 2008; 9: 139-150Crossref PubMed Scopus (2440) Google Scholar). S1P acts as an agonist at specific G protein-coupled receptors, termed S1P1–5, to regulate cell growth, migration, and cell-cell contacts and thereby modulates lymphocyte emigration from lymphatic tissues, angiogenesis, vascular barrier function, tissue homeostasis, and inflammation (6Maceyka M. Harikumar K.B. Milstien S. Spiegel S. Sphingosine 1-phosphate signaling and its role in disease.Trends Cell Biol. 2012; 22: 50-60Abstract Full Text Full Text PDF PubMed Scopus (748) Google Scholar, 7Blaho V.A. Hla T. An update on the biology of sphingosine 1-phosphate receptors.J. Lipid Res. 2014; Abstract Full Text Full Text PDF PubMed Scopus (341) Google Scholar). In addition, several intracellular activities of S1P have been described recently, where this mediator was delivered by SphK1 or SphK2 directly to a target protein, for example histone deacetylases (HDACs), tumor necrosis factor receptor-associated factor-2, mitochondrial prohibitin-2, or β-site amyloid precursor protein cleaving enzyme-1 [reviewed in Maceyka et al. (6Maceyka M. Harikumar K.B. Milstien S. Spiegel S. Sphingosine 1-phosphate signaling and its role in disease.Trends Cell Biol. 2012; 22: 50-60Abstract Full Text Full Text PDF PubMed Scopus (748) Google Scholar)]. In contrast to these apparently direct interactions of SphKs with S1P target proteins, intracellularly generated S1P has to be transported across the plasma membrane to be able to activate its specific G protein-coupled receptors (8Takabe K. Spiegel S. Export of sphingosine 1-phosphate and cancer progression.J. Lipid Res. 2014; Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). Among the transporter proteins that have been shown to export S1P, there are several members of the ABC transporter family such as the multidrug resistance-related protein Abcc1, the cholesterol transporter Abca1, and the breast cancer resistance protein Abcg2 (9Mitra P. Oskeritzian C.A. Payne S.G. Beaven M.A. Milstien S. Spiegel S. Role of ABCC1 in export of sphingosine 1-phosphate from mast cells.Proc. Natl. Acad. Sci. USA. 2006; 103: 16394-16399Crossref PubMed Scopus (348) Google Scholar, 10Sato K. Malchinkhuu E. Horiuchi Y. Mogi C. Tomura H. Tosaka M. Yoshimoto Y. Kuwabara A. Okajima F. Critical role of ABCA1 transporter in sphingosine 1-phosphate release from astrocytes.J. Neurochem. 2007; 103: 2610-2619PubMed Google Scholar, 11Takabe K. Kim R.H. Allegood J.C. Mitra P. Ramachandran S. Nagahashi M. Harikumar K.B. Hait N.C. Milstien S. Spiegel S. Estradiol induces export of sphingosine 1-phosphate from breast cancer cells via ABCC1 and ABCG2.J. Biol. Chem. 2010; 285: 10477-10486Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar). While these ABC transporters either do not regulate plasma concentrations of S1P or are redundant with respect to this activity (12Lee Y-M. Venkataraman K. Hwang S-I. Han D.K. Hla T. A novel method to quantify sphingosine 1-phosphate by immobilized metal affinity chromatography (IMAC).Prostaglandins Other Lipid Mediat. 2007; 84: 154-162Crossref PubMed Scopus (99) Google Scholar), plasma S1P is regulated by the non-ABC transporter-related transport protein spinster-2, which is probably a specific S1P transporter (13Fukuhara S. Simmons S. Kawamura S. Inoue A. Orba Y. Tokudome T. Sunden Y. Arai Y. Moriwaki K. Ishida J. et al.The sphingosine 1-phosphate transporter Spns2 expressed on endothelial cells regulates lymphocyte trafficking in mice.J. Clin. Invest. 2012; 122: 1416-1426Crossref PubMed Scopus (237) Google Scholar, 14Hisano Y. Kobayashi N. Yamaguchi A. Nishi T. Mouse SPNS2 functions as a sphingosine 1-phosphate transporter in vascular endothelial cells.PLoS ONE. 2012; 7: e38941Crossref PubMed Scopus (166) Google Scholar). While the role of ABC transporters in chemoresistance of cancers is widely recognized (1Fodale V. Pierobon M. Liotta L. Petricoin E. Mechanism of cell adaptation: when and how do cancer cells develop chemoresistance?.Cancer J. 2011; 17: 89-95Crossref PubMed Scopus (150) Google Scholar, 8Takabe K. Spiegel S. Export of sphingosine 1-phosphate and cancer progression.J. Lipid Res. 2014; Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 15Yu M. Ocana A. Tannock I.F. Reversal of ATP-binding cassette drug transporter activity to modulate chemoresistance: why has it failed to provide clinical benefit?.Cancer Metastasis Rev. 2013; 32: 211-227Crossref PubMed Scopus (117) Google Scholar), the role of the enzymes that catalyze S1P formation and degradation in cancer growth and survival appears to be less clear. SphK1 is upregulated in many cancers, and cancer cells might have a "non-oncogene addiction" for this enzyme (16Pyne N.J. Pyne S. Sphingosine 1-phosphate and cancer.Nat. Rev. Cancer. 2010; 10: 489-503Crossref PubMed Scopus (672) Google Scholar); however, new highly potent SphK1 and SphK1/2 inhibitors failed to inhibit cancer cell proliferation and growth of tumor xenografts in mice (17Schnute M.E. McReynolds M.D. Kasten T. Yates M. Jerome G. Rains J.W. Hall T. Chrencik J. Kraus M. Cronin C.N. et al.Modulation of cellular S1P levels with a novel, potent and specific inhibitor of sphingosine kinase-1.Biochem. J. 2012; 444: 79-88Crossref PubMed Scopus (209) Google Scholar, 18Rex K. Jeffries S. Brown M.L. Carlson T. Coxon A. Fajardo F. Frank B. Gustin D. Kamb A. Kassner P.D. et al.Sphingosine kinase activity is not required for tumor cell viability.PLoS ONE. 2013; 8: e68328Crossref PubMed Scopus (56) Google Scholar). Recently, also S1P lyase, which is an endoplasmic reticulum (ER) resident enzyme that cleaves S1P irreversibly and catalyzes the ultimate step in sphingolipid catabolism, has been connected with cancer growth and chemoresistance (19Colié S. van Veldhoven P.P. Kedjouar B. Bedia C. Albinet V. Sorli S-C. Garcia V. Djavaheri-Mergny M. Bauvy C. Codogno P. et al.Disruption of sphingosine 1-phosphate lyase confers resistance to chemotherapy and promotes oncogenesis through Bcl-2/Bcl-xL upregulation.Cancer Res. 2009; 69: 9346-9353Crossref PubMed Scopus (93) Google Scholar, 20Aguilar A. Saba J.D. Truth and consequences of sphingosine 1-phosphate lyase.Adv. Biol. Regul. 2012; 52: 17-30Crossref PubMed Scopus (55) Google Scholar, 21Brizuela L. Ader I. Mazerolles C. Bocquet M. Malavaud B. Cuvillier O. First evidence of sphingosine 1-phosphate lyase protein expression and activity downregulation in human neoplasm: implication for resistance to therapeutics in prostate cancer.Mol. Cancer Ther. 2012; 11: 1841-1851Crossref PubMed Scopus (56) Google Scholar). Early studies have shown that overexpression of S1P lyase rendered the cells sensitive to apoptosis induced by serum deprivation or chemotherapeutic agents, while siRNA-induced knockdown diminished apoptosis at baseline and in response to chemotherapy (22Reiss U. Oskouian B. Zhou J. Gupta V. Sooriyakumaran P. Kelly S. Wang E. Merrill A.H. Saba J.D. Sphingosine-phosphate lyase enhances stress-induced ceramide generation and apoptosis.J. Biol. Chem. 2004; 279: 1281-1290Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, 23Min J. van Veldhoven P.P. Zhang L. Hanigan M.H. Alexander H. Alexander S. Sphingosine 1-phosphate lyase regulates sensitivity of human cells to select chemotherapy drugs in a p38-dependent manner.Mol. Cancer Res. 2005; 3: 287-296Crossref PubMed Scopus (60) Google Scholar, 24Oskouian B. Sooriyakumaran P. Borowsky A.D. Crans A. Dillard-Telm L. Tam Y.Y. Bandhuvula P. Saba J.D. Sphingosine 1-phosphate lyase potentiates apoptosis via p53- and p38-dependent pathways and is down-regulated in colon cancer.Proc. Natl. Acad. Sci. USA. 2006; 103: 17384-17389Crossref PubMed Scopus (169) Google Scholar). Recent studies show that S1P lyase is downregulated in human colon cancers (24Oskouian B. Sooriyakumaran P. Borowsky A.D. Crans A. Dillard-Telm L. Tam Y.Y. Bandhuvula P. Saba J.D. Sphingosine 1-phosphate lyase potentiates apoptosis via p53- and p38-dependent pathways and is down-regulated in colon cancer.Proc. Natl. Acad. Sci. USA. 2006; 103: 17384-17389Crossref PubMed Scopus (169) Google Scholar), human melanoma cell lines (19Colié S. van Veldhoven P.P. Kedjouar B. Bedia C. Albinet V. Sorli S-C. Garcia V. Djavaheri-Mergny M. Bauvy C. Codogno P. et al.Disruption of sphingosine 1-phosphate lyase confers resistance to chemotherapy and promotes oncogenesis through Bcl-2/Bcl-xL upregulation.Cancer Res. 2009; 69: 9346-9353Crossref PubMed Scopus (93) Google Scholar), and human prostate cancers (21Brizuela L. Ader I. Mazerolles C. Bocquet M. Malavaud B. Cuvillier O. First evidence of sphingosine 1-phosphate lyase protein expression and activity downregulation in human neoplasm: implication for resistance to therapeutics in prostate cancer.Mol. Cancer Ther. 2012; 11: 1841-1851Crossref PubMed Scopus (56) Google Scholar). Fibroblasts lacking S1P lyase were able to form colonies in soft agar and to induce tumors in immunocompromised mice (19Colié S. van Veldhoven P.P. Kedjouar B. Bedia C. Albinet V. Sorli S-C. Garcia V. Djavaheri-Mergny M. Bauvy C. Codogno P. et al.Disruption of sphingosine 1-phosphate lyase confers resistance to chemotherapy and promotes oncogenesis through Bcl-2/Bcl-xL upregulation.Cancer Res. 2009; 69: 9346-9353Crossref PubMed Scopus (93) Google Scholar). In human prostate cancers, S1P lyase expression and activity was inversely correlated with clinical malignancy scores, and cell death of prostate cancer cell lines, induced by irradiation or chemotherapy, was reduced by S1P lyase knockdown and potentiated by S1P lyase overexpression (21Brizuela L. Ader I. Mazerolles C. Bocquet M. Malavaud B. Cuvillier O. First evidence of sphingosine 1-phosphate lyase protein expression and activity downregulation in human neoplasm: implication for resistance to therapeutics in prostate cancer.Mol. Cancer Ther. 2012; 11: 1841-1851Crossref PubMed Scopus (56) Google Scholar). Although a low expression of S1P lyase thus appears to be favorable for proliferation and survival of cancer cells, the presence of the enzyme is essential for development and survival of the mammalian organism as a whole. Thus, a full knockout of S1P lyase in the mouse induced growth retardation and early death after a few weeks, accompanied by severe organ damage, immunosuppression, a major disturbance of lipid homeostasis, and a proinflammatory phenotype [for review, see Aguilar and Saba (20Aguilar A. Saba J.D. Truth and consequences of sphingosine 1-phosphate lyase.Adv. Biol. Regul. 2012; 52: 17-30Crossref PubMed Scopus (55) Google Scholar)]. On the other hand, mouse embryonic fibroblasts (MEFs) from S1P lyase-deficient mice (Sgpl1−/−-MEFs) proliferated without retardation and, in the absence of serum, even better than MEFs from wild-type mice (19Colié S. van Veldhoven P.P. Kedjouar B. Bedia C. Albinet V. Sorli S-C. Garcia V. Djavaheri-Mergny M. Bauvy C. Codogno P. et al.Disruption of sphingosine 1-phosphate lyase confers resistance to chemotherapy and promotes oncogenesis through Bcl-2/Bcl-xL upregulation.Cancer Res. 2009; 69: 9346-9353Crossref PubMed Scopus (93) Google Scholar). The resistance of Sgpl1−/−-MEFs to apoptosis induced by doxorubicin and etoposide was ascribed to upregulation of the antiapoptotic proteins Bcl-2 and Bcl-xL because a combined knockdown of both proteins with siRNA improved the sensitivity of the cells (19Colié S. van Veldhoven P.P. Kedjouar B. Bedia C. Albinet V. Sorli S-C. Garcia V. Djavaheri-Mergny M. Bauvy C. Codogno P. et al.Disruption of sphingosine 1-phosphate lyase confers resistance to chemotherapy and promotes oncogenesis through Bcl-2/Bcl-xL upregulation.Cancer Res. 2009; 69: 9346-9353Crossref PubMed Scopus (93) Google Scholar). In our own studies on these S1P lyase-deficient fibroblasts, we observed that S1P accumulated by ∼5-fold compared with wild-type cells when whole cells were extracted and ∼50-fold in nuclear preparations (25Claas R.F. Ter Braak M. Hegen B. Hardel V. Angioni C. Schmidt H. Jakobs K.H. van Veldhoven P.P. Meyer zu Heringdorf D. Enhanced Ca2+ storage in sphingosine 1-phosphate lyase-deficient fibroblasts.Cell. Signal. 2010; 22: 476-483Crossref PubMed Scopus (22) Google Scholar, 26Ihlefeld K. Claas R.F. Koch A. Pfeilschifter J.M. Meyer zu Heringdorf D. Evidence for a link between histone deacetylation and Ca2+ homoeostasis in sphingosine 1-phosphate lyase-deficient fibroblasts.Biochem. J. 2012; 447: 457-464Crossref PubMed Scopus (31) Google Scholar). Furthermore, we observed a reduced HDAC activity and downregulation of class I HDACs, which contributed to the disturbed Ca2+ homeostasis with enhanced Ca2+ storage and elevated basal intracellular free Ca2+ concentration ([Ca2+]i ) in these cells (26Ihlefeld K. Claas R.F. Koch A. Pfeilschifter J.M. Meyer zu Heringdorf D. Evidence for a link between histone deacetylation and Ca2+ homoeostasis in sphingosine 1-phosphate lyase-deficient fibroblasts.Biochem. J. 2012; 447: 457-464Crossref PubMed Scopus (31) Google Scholar). The starting point of our present study was the hypothesis that this accumulation of S1P in S1P lyase-deficient MEFs may induce counterregulatory mechanisms such as upregulation of S1P secretion, which would then keep cytosolic S1P at a normal level while nuclear pools of S1P probably have no access to the export mechanisms. Therefore, we studied the expression and functionality of the transporters that have been implicated in S1P export, i.e., spinster-2, Abcc1, Abca1, and Abcg2, and also of the multidrug resistance protein Abcb1, which is known to be regulated by SphK and S1P (27Pilorget A. Demeule M. Barakat S. Marvaldi J. Luis J. Béliveau R. Modulation of P-glycoprotein function by sphingosine kinase-1 in brain endothelial cells.J. Neurochem. 2007; 100: 1203-1210Crossref PubMed Scopus (44) Google Scholar). We demonstrate that several of the transporters were upregulated in Sgpl1−/−-MEFs and caused a sequestration of the ABC transporter substrates fluo-4 and doxorubicin. Furthermore, we show that the compartmentalization of doxorubicin contributes significantly to the chemoresistance of these cells. These results link S1P lyase to the regulation of multidrug transporters and suggest that this activity, in addition to S1P's ability to interfere with apoptotic signaling pathways, plays an important role in cancer cell chemoresistance. Fluo-4/AM, tetramethylrhodamine, ER-Tracker Blue-White DPX, LysoTracker Red DND-99, and Hoechst 33342 were obtained from Molecular Probes/Invitrogen (Invitrogen GmbH, Karlsruhe, Germany). Doxorubicin, MK571, probenecid, verapamil, staurosporine, and fatty acid-free BSA were purchased from Sigma-Aldrich Chemie GmbH (Taufkirchen, Germany). Trichostatin A (TSA) was from Calbiochem/Merck Millipore (Darmstadt, Germany), and zosuquidar was from Selleck Chemicals LLC (Houston, TX). All other chemicals were from previously described sources (25Claas R.F. Ter Braak M. Hegen B. Hardel V. Angioni C. Schmidt H. Jakobs K.H. van Veldhoven P.P. Meyer zu Heringdorf D. Enhanced Ca2+ storage in sphingosine 1-phosphate lyase-deficient fibroblasts.Cell. Signal. 2010; 22: 476-483Crossref PubMed Scopus (22) Google Scholar, 26Ihlefeld K. Claas R.F. Koch A. Pfeilschifter J.M. Meyer zu Heringdorf D. Evidence for a link between histone deacetylation and Ca2+ homoeostasis in sphingosine 1-phosphate lyase-deficient fibroblasts.Biochem. J. 2012; 447: 457-464Crossref PubMed Scopus (31) Google Scholar). Embryonic fibroblasts from S1P lyase-deficient and corresponding wild-type mice had been prepared as described previously (25Claas R.F. Ter Braak M. Hegen B. Hardel V. Angioni C. Schmidt H. Jakobs K.H. van Veldhoven P.P. Meyer zu Heringdorf D. Enhanced Ca2+ storage in sphingosine 1-phosphate lyase-deficient fibroblasts.Cell. Signal. 2010; 22: 476-483Crossref PubMed Scopus (22) Google Scholar). The cells were cultured in DMEM/F12 supplemented with 100 U/ml penicillin G, 0.1 mg/ml streptomycin, and 10% FCS in a humidified atmosphere of 5% CO2/95% air at 37°C. If not stated otherwise, the cells were kept in serum-free medium overnight before experiments. The cells were seeded onto 3.5 cm dishes and grown to near confluence. They were kept in serum-free medium supplemented with 1 mg/ml fatty acid-free BSA in the absence or presence of 10 µM sphingosine for 16 h. Thereafter, the cellular supernatants (1.5 ml) were collected and centrifuged for 10 min at 1,800 × g and 4°C. The supernatants were transferred into fresh tubes and supplemented with 1.2 ml methanol containing 20 ng/ml d-erythro-C17-sphingosine and 20 ng/ml d-erythro-C17-S1P (Avanti Polar Lipids Inc., Alabaster, AL) as internal standards. Then 35 µl 1 M HCl, 70 µl 10% KCl, and 2 ml chloroform were added, and after thorough mixing and centrifugation, the organic phase was collected. The aqueous phase was reextracted two times with chloroform, and the organic phases were combined and dried down. The lipids were redissolved in 200 µl DMSO containing 2% HCl, and LC-MS/MS was performed as previously described (28Schmidt H. Schmidt R. Geisslinger G. LC-MS/MS-analysis of sphingosine 1-phosphate and related compounds in plasma samples.Prostaglandins Other Lipid Mediat. 2006; 81: 162-170Crossref PubMed Scopus (52) Google Scholar). The cell pellets were washed, scraped into lysis buffer, and subjected to protein measurements. The cells were seeded onto 3.5 cm dishes and grown to near confluence. Before experiments, they were kept for 16 h either in serum-free medium supplemented with 10 mg/ml fatty acid-free BSA or in medium containing 10% FCS. Labeling was performed in the respective media for 2 h with 0.5 µCi/ml [3H]sphingosine. Then, the cells were washed twice and incubated for a further 4 h either in serum-free medium supplemented with 10 mg/ml fatty acid-free BSA or in medium containing 10% FCS. Thereafter, the cellular supernatants (1 ml) were collected, and 1 ml methanol, 70 µl 10% KCl, 35 µl 1 M HCl, and 2 ml chloroform were added. Cell monolayers were washed with ice-cold PBS and scraped into 1 ml methanol. The dishes were washed with 1 ml methanol, and 1.6 ml of high salt solution (0.74% KCl, 0.04% CaCl2, 0.034% MgCl2), 35 µl 1 M HCl, and 2 ml chloroform were added. Lipid extraction was performed as described for the LC-MS/MS measurements. The dried samples were redissolved in 50 µl methanol and separated by TLC with 1-butanol:acetic acid:water 3:1:1. Areas containing S1P and sphingosine, respectively, were identified with nonradioactive standard samples and scraped off the TLC plates, and radioactivity was quantified by liquid scintillation counting. Separate dishes were used for protein measurements. For microscopic analysis, the cells were cultured on 8-well chambered coverslides (µ-slide; ibidi GmbH, Martinsried, Germany) coated with poly-l-lysine. If not stated otherwise, the cells were washed with HBSS and kept in HBSS at room temperature during the measurements. Confocal laser scanning microscopy was performed with a Zeiss LSM510 Meta system equipped with an inverted Observer Z1 microscope and a Plan-Apochromat 63×/1.4 oil immersion objective (Carl Zeiss MicroImaging GmbH, Göttingen, Germany). The following excitation (ex) laser lines and emission (em) filter sets were used: fluo-4, ex 488 nm, em 505 nm long pass filter or 505–530 nm band pass filter (when measured in combination with tetramethylrhodamine); tetramethylrhodamine and LysoTracker Red DND-99, ex 543 nm, em 560 nm long pass filter; ER-Tracker Blue-White DPX, ex 364 nm, em 475 nm long pass filter; Hoechst 33342, ex 405 nm, em 420–480 nm band pass filter; and doxorubicin, ex 488 nm, em 575–630 nm band pass filter. Simultaneous staining with more than one dye was analyzed in multitracking mode. The subcellular distribution of Abcb1 was analyzed by immunocytochemistry. Cells grown on 8-well chambered coverslides were fixed with formaldehyde and stained with anti-p-glycoprotein antibody (Sigma-Aldrich Chemie GmbH) followed by Alexa-Fluor 555-conjugated anti-mouse antibody (Invitrogen GmbH). The fluorescence was monitored by confocal microscopy as described above using the 543 nm excitation laser line and a 575–630 nm emission band pass filter. mRNA was isolated from serum-starved MEFs with TRIZOL (Sigma-Aldrich Chemie GmbH). cDNA was prepared with the RevertAid first strand cDNA synthesis kit (Fermentas, St. Leon-Rot, Germany). Real-time PCR was performed with the Applied Biosystems 7500 Fast Real-Time PCR System. Probes, primers, and the reporter dyes 6-FAM and VIC were from Applied Biosystems (Darmstadt, Germany). The cycling conditions were 95°C for 15 min (1 cycle), followed by 95°C for 15 s and 60°C for 1 min (40 cycles). mRNA expression levels were analyzed by the ΔΔCt method with GAPDH as reference. Cell lysates were separated by SDS gel electrophoresis and blotted onto polyvinylidene difluoride membranes. Blots were stained with antibodies directed against Abcc1 (Abcam, Cambridge, UK), caspase-3 (Cell Signaling Technology, Danvers, MA), or β-actin (Santa Cruz Biotechnology Inc., Heidelberg, Germany) and analyzed with HRP-conjugated secondary antibodies using the ECL system (GE Healthcare, Freiburg, Germany). In the first set of experiments, Sgpl1+/+- and Sgpl1−/−-MEFs were seeded onto 96-well plates and grown to near confluence. The cells were incubated with doxorubicin, staurosporine, or vehicle for 16 h in serum-free medium, and cell viability was analyzed with the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay according to the manufacturer's instructions (CellTiter 96 NonRadioactive Cell Proliferation Assay; Promega, Mannheim, Germany). In the second set of experiments, MEFs were seeded onto 24-well plates, grown to near confluence, and pretreated with ABC transporter inhibitors for 1 h before addition of the chemotherapeutic agents. Cell viability was analyzed with the sodium 2,3,-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)-carbonyl]-2H-tetrazolium (XTT) assay (Cell Proliferation Assay XTT; AppliChem, Darmstadt, Germany). Averaged data are means ± SEM from the indicated number (n) of independent experiments or means ± SD from a representative experiment performed with n replicates. Graphical presentation and statistical analysis were performed with Prism-5 (GraphPad Software, San Diego, CA). Microscopic images are representative for at least three similar experiments and were edited using the LSM Image Browser or the ZEN software (http://www.zeiss.com/micro). Because S1P accumulated in S1P lyase-deficient MEFs, we hypothesized that the cells would try to counteract this by upregulating S1P secretion. Therefore, we analyzed the expression of the multidrug transporters Abcc1 (= MRP1), Abca1, and Abcg2, which all have been associated with S1P extrusion, of Abcb1 (= MDR1 or p-glycoprotein), which is known to be regulated by S1P metabolism (27Pilorget A. Demeule M. Barakat S. Marvaldi J. Luis J. Béliveau R. Modulation of P-glycoprotein function by sphingosine kinase-1 in brain endothelial cells.J. Neurochem. 2007; 100: 1203-1210Crossref PubMed Scopus (44) Google Scholar), and of spinster-2, which appears to be a specific S1P transporter (13Fukuhara S. Simmons S. Kawamura S. Inoue A. Orba Y. Tokudome T. Sunden Y. Arai Y. Moriwaki K. Ishida J. et al.The sphingosine 1-phosphate transporter Spns2 expressed on endothelial cells regulates lymphocyte trafficking in mice.J. Clin. Invest. 2012; 122: 1416-1426Crossref PubMed Scopus (237) Google Scholar, 14Hisano Y. Kobayashi N. Yamaguchi A. Nishi T. Mouse SPNS2 functions as a sphingosine 1-phosphate transporter in vascular endothelial cells.PLoS ONE. 2012; 7: e38941Crossref PubMed Scopus (166) Google Scholar). Indeed, the mRNA levels of Abcc1 and Abca1 were upregulated by ∼2- to 3-fold in Sgpl1−/−-MEFs, while Abcg2 remained unaltered (Fig. 1A). Of the two transcripts of Abcb1, Abcb1a was strongly induced by ∼10-fold, while Abcb1b was upregulated by ∼2-fold (Fig. 1A). Spinster-2 mRNA was induced by ∼3-fold in Sgpl1−/−-MEFs (Fig. 1A). On the protein level, both the unglycosylated form at ∼130 kDa and the glycosylated mature form at ∼170 kDa of Abcc1 were upregulated in Sgpl1−/−-MEFs (Fig. 1B). The expression of the mature form was quantified by densitometry and found to be enhanced by ∼2-fold (Fig. 1B). Because it is known that HDAC inhibitors can upregulate multidrug resistance transporters in tumor cells, and because HDAC activity and expression of class I HDACs were decreased in Sgpl1−/−-MEFs, we analyzed whether a treatment with the pan-HDAC inhibitor TSA induced an upregulation of the transporters in wild-type cells. However, TSA did not upregulate Abcc1, Abcb1, or Abca1 in either Sgpl1+/+- or Sgpl1−/−-MEFs (data not shown). In contrast, TSA strongly induced the expression of spinster-2 by ∼10-fold in both Sgpl1+/+- and Sgpl1−/−-MEFs (Fig. 1A). Spinster-2 expression thus appears to be regulated by histone acetylation. Because the transporters that could potentially transport S1P were upregulated in Sgpl1−/−-MEFs, we analyzed whether S1P was elevated in the supernatants of these cells. However, although total S1P levels were ∼5-fold higher in S1P lyase-deficient MEFs (25Claas R.F. Ter Braak M. Hegen B. Hardel V. Angioni C. Schmidt H. Jakobs K.H. van Veldho
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