The number of patients eligible for allogeneic stem cell transplantation (allo-HSCT) has increased because of improvements in transplantation procedures. Among long-term survivors of allo-HSCT, chronic kidney disease (CKD) is a major cause of morbidity. We retrospectively analyzed the clinical data of 106 consecutive patients with a median age of 43 years (range, 17 to 73) who had undergone allo-HSCT at our institution between January 2001 and September 2009. Patients who died within 5 years after transplantation or had CKD at the time of transplantation were excluded from study. CKD was defined as a persistent decrease in the estimated glomerular filtration rate to below 60 mL/min/1.73 m2. CKD occurred in 32 patients (30.2%) at a median time of 55 months after transplantation. Three patients required maintenance hemodialysis. In multivariate analysis older age at the time of transplantation (hazard ratio [HR], 1.07/year; 95% confidence interval [CI], 1.02 to 1.13) and a history of acute kidney injury (AKI) within 100 days after transplantation (HR, 6.30; 95% CI, 2.21 to 17.9) were significant risk factors for CKD. Conditioning regimen, stem cell source, or the presence of acute/chronic GVHD was not significantly associated with CKD in this study. Patients with CKD had a lower overall survival rate (HR, 4.11; 95% CI, 1.3 to 13.0) than patients without CKD. Careful monitoring of renal function is required for long-term survivors after allo-HSCT, especially in patients who have experienced AKI and in older patients.
TAFRO syndrome is a newly defined disease entity which is characterized by thrombocytopenia, anasarca, myelofibrosis, renal dysfunction, and organomegaly. A histological pattern of multiple lymphadenopathy of atypical Castleman's disease (CD) is also an important characteristic. A 48-year-old man was referred to our hospital with fever, asthenia, bilateral pleural effusion, ascites, generalized edema, dyspnea, hypoalbuminemia, severe thrombocytopenia, anemia, renal failure and proteinuria, whereas bacterial culture and serological and PCR tests for various viruses were all negative. A CT scan showed multiple lymphadenopathy and tissue sampling of inguinal lymph nodes showed a compatible histology with plasma cell type CD. A diagnosis of TAFRO syndrome was made. Ten days after hospitalization, sudden cardiac insufficiency and anuria developed. Despite glucocorticoid pulse therapy, tocilizumab and plasmapheresis, clinical and laboratory features did not improve. On the 34th hospital day, we started rituximab. His general condition started to improve in several days, and by one month later anasarca had improved drastically. Thrombocytopenia and renal function gradually improved and finally normalized. Cardiac motion also improved. This is the first report of a TAFRO syndrome patient with cardiomyopathy, who was successfully treated with rituximab.
Sphingomyelin (SM) synthase has been assumed to be involved in both cell death and survival by regulating pro-apoptotic mediator ceramide and pro-survival mediator diacylglycerol. However, its precise functions are ambiguous due to the lack of molecular cloning of SM synthase gene(s). We isolated WR19L/Fas-SM(-) mouse lymphoid cells, which show a defect of SM at the plasma membrane due to the lack of SM synthase activity and resistance to cell death induced by an SM-directed cytolytic protein lysenin. WR19L/Fas-SM(-) cells were also highly susceptible to methyl-β-cyclodextrin (MβCD) as compared with the WR19L/Fas-SM(+) cells, which are capable of SM synthesis. By expression cloning method using WR19L/Fas-SM(-) cells and MβCD-based selection, we have succeeded in cloning of a human cDNA responsible for SM synthase activity. The cDNA encodes a peptide of 413 amino acids named SMS1 (putative molecular mass, 48.6 kDa), which contains a sterile α motif domain near the N-terminal region and four predicted transmembrane domains. WR19L/Fas-SM(-) cells expressing SMS1 cDNA (WR19L/Fas-SMS1) restored the resistance against MβCD, the accumulation of SM at the plasma membrane, and SM synthesis by transferring phosphocholine from phosphatidylcholine to ceramide. Furthermore, WR19L/Fas-SMS1 cells, as well as WR19L/Fas-SM(-) cells supplemented with exogenous SM, restored cell growth ability in serum-free conditions, where the growth of WR19L/Fas-SM(-) cells was severely inhibited. The results suggest that SMS1 is responsible for SM synthase activity in mammalian cells and plays a critical role in cell growth of mouse lymphoid cells. Sphingomyelin (SM) synthase has been assumed to be involved in both cell death and survival by regulating pro-apoptotic mediator ceramide and pro-survival mediator diacylglycerol. However, its precise functions are ambiguous due to the lack of molecular cloning of SM synthase gene(s). We isolated WR19L/Fas-SM(-) mouse lymphoid cells, which show a defect of SM at the plasma membrane due to the lack of SM synthase activity and resistance to cell death induced by an SM-directed cytolytic protein lysenin. WR19L/Fas-SM(-) cells were also highly susceptible to methyl-β-cyclodextrin (MβCD) as compared with the WR19L/Fas-SM(+) cells, which are capable of SM synthesis. By expression cloning method using WR19L/Fas-SM(-) cells and MβCD-based selection, we have succeeded in cloning of a human cDNA responsible for SM synthase activity. The cDNA encodes a peptide of 413 amino acids named SMS1 (putative molecular mass, 48.6 kDa), which contains a sterile α motif domain near the N-terminal region and four predicted transmembrane domains. WR19L/Fas-SM(-) cells expressing SMS1 cDNA (WR19L/Fas-SMS1) restored the resistance against MβCD, the accumulation of SM at the plasma membrane, and SM synthesis by transferring phosphocholine from phosphatidylcholine to ceramide. Furthermore, WR19L/Fas-SMS1 cells, as well as WR19L/Fas-SM(-) cells supplemented with exogenous SM, restored cell growth ability in serum-free conditions, where the growth of WR19L/Fas-SM(-) cells was severely inhibited. The results suggest that SMS1 is responsible for SM synthase activity in mammalian cells and plays a critical role in cell growth of mouse lymphoid cells. Diverse kinds of phospho- and glycerolipids such as diacylglycerol (DAG), 1The abbreviations used are: DAG, diacylglycerol; SM, sphingomyelin; PC, phosphatidylcholine; MβCD, methyl-β-cyclodextrin; SAM, sterile α motif; WST-8, 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium; FBS, fetal bovine serum; FACS, fluorescence-activated cell sorter; MBP, maltose-binding protein; NBD, 12-(N-methyl-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)). 1The abbreviations used are: DAG, diacylglycerol; SM, sphingomyelin; PC, phosphatidylcholine; MβCD, methyl-β-cyclodextrin; SAM, sterile α motif; WST-8, 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium; FBS, fetal bovine serum; FACS, fluorescence-activated cell sorter; MBP, maltose-binding protein; NBD, 12-(N-methyl-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)). inositol phosphatides, and phosphatidic acid are recognized as bioactive molecules in cell growth and survival (1English D. Cell. Signal. 1996; 8: 341-347Google Scholar, 2Majerus P.W. Annu. Rev. Biochem. 1992; 61: 225-250Google Scholar). Sphingolipid ceramide has recently emerged as a signal mediator of cell functions including apoptosis, differentiation, and secretion (3Pettus B.J. Chalfant C.E. Hannun Y.A. Biochim. Biophys. Acta. 2002; 1585: 114-125Google Scholar). Various stresses such as ultraviolet, irradiation, heat shock, hypoxia, and biological factors such as tumor necrosis factor-α, interferon-γ, and Fas antibody require ceramide generation to execute apoptosis, suggesting the implications of SM as a source of ceramide generation in the induction of cell death (4Okazaki T. Kondo T. Kitano T. Tashima M. Cell. Signal. 1998; 10: 685-692Google Scholar, 5Hannun Y.A. J. Biol. Chem. 1994; 269: 3125-3128Google Scholar). It was reported that SM dose-dependently inhibits both deoxycholate-induced apoptosis and subsequent hyper-proliferation in colon epithelial cells (6Moschetta A. Portincasa P. van Erpecum K.J. Debellis L. Vanberge-Henegouwen G.P. Palasciano G. Dig. Dis. Sci. 2003; 48: 1094-1101Google Scholar) and decreases the number of aberrant crypts of colon (7Dillehay D.L. Webb S.K. Schmelz E.M. Merrill Jr., A.H. J. Nutr. 1994; 124: 615-620Google Scholar), suggesting the implications of SM in cell death and growth.SM is produced by SM synthase, which is thought to be the only enzyme to synthesize SM in mammalian cells (8Voelker D.R. Kennedy E.P. Biochemistry. 1982; 21: 2753-2759Google Scholar). The enzyme catalyzes the reaction in which phosphocholine moiety is transferred from phosphatidylcholine (PC) to ceramide. Thus, the activation of SM synthase subsequently increases the levels of DAG and decreases ceramide at the same time (8Voelker D.R. Kennedy E.P. Biochemistry. 1982; 21: 2753-2759Google Scholar). DAG is an important signaling molecule for cell growth through protein kinase C activation (9Hampton R.Y. Morand O.H. Science. 1989; 2461050Google Scholar, 10Pagano R.E. Trends Biochem. Sci. 1988; 13: 202-205Google Scholar, 11Moscat J. Cornet M.E. Diaz-Meco M.T. Larrodera P. Lopez-Alanon D. Lopez-Barahona M. Biochem. Soc. Trans. 1989; 17: 988-991Google Scholar, 12Lucas L. del Peso L. Rodriguez P. Penalva V. Lacal J.C. Oncogene. 2000; 19: 431-437Google Scholar) and acts competitively against ceramide-induced apoptosis (4Okazaki T. Kondo T. Kitano T. Tashima M. Cell. Signal. 1998; 10: 685-692Google Scholar, 13Hannun Y.A. Bell R.M. Science. 1989; 243: 500-507Google Scholar). It has been reported that after thioacetamide-induced injury, the SM/PC ratio significantly increased in microsomal fraction from liver, suggesting the involvement of SM synthase in tissue recovery (14Miro-Obradors M.J. Osada J. Aylagas H. Sanchez-Vegazo I. Palacios-Alaiz E. Carcinogenesis. 1993; 14: 941-946Google Scholar). In cerebellar astrocytes, the level of ceramide is rapidly down-regulated by basic fibroblast growth factor via activating SM synthase (15Riboni L. Tettamanti G. Viani P. Cerebellum. 2002; 1: 129-135Google Scholar). In SV40-transformed lung fibroblasts, SM synthase regulates the levels of ceramide and DAG in an opposite direction (16Luberto C. Hannun Y.A. J. Biol. Chem. 1998; 273: 14550-14559Google Scholar). We recently reported that SM synthase was activated to inhibit ceramide generation in IL-2-induced proliferation of natural killer cells, 2Y. Taguchi, T. Kondo, M. Watanabe, Y. Kozutumi, and T. Okazaki, submitted for publication. 2Y. Taguchi, T. Kondo, M. Watanabe, Y. Kozutumi, and T. Okazaki, submitted for publication. whereas the activity in nucleus was inhibited with ceramide generation in Fas-induced T cell apoptosis (17Watanabe M. Kitano T. Kondo T. Yabu T. Taguchi Y. Tashima M. Umehara H. Domae N. Uchiyama T. Okazaki T. Cancer Res. 2004; 64: 1-8Google Scholar). We also showed its in vivo implication that the level of ceramide was decreased via activation of SM synthase in chemotherapy-resistant blast cells obtained from refractory leukemia patients than in chemotherapy-sensitive leukemic blasts (18Itoh M. Kitano T. Watanabe M. Kondo T. Yabu T. Taguchi Y. Iwai K. Tashima M. Uchiyama T. Okazaki T. Clin. Cancer Res. 2003; 9: 415-423Google Scholar). Thus, SM synthase is assumed to play an important role in cell death and survival, in vitro as well as in vivo.We previously proposed the "SM cycle," a pathway that consisted of SM synthase and sphingomyelinase as a novel biological system to regulate the cellular level of ceramide for cell death and differentiation (19Okazaki T. Bell R.M. Hannun Y.A. J. Biol. Chem. 1989; 264: 19076-19080Google Scholar). In contrast to the studies of the acid and neutral sphingomyelinases in cell death (20Chatterjee S. Chem. Phys. Lipids. 1999; 102: 79-96Google Scholar, 21Cremesti A.E. Goni F.M. Kolesnick R. FEBS Lett. 2002; 531: 47-53Google Scholar), the biological implication of SM synthase has not been elucidated due to the lack of molecular cloning of its responsible gene(s). We recently found mouse lymphoid cell variants designated WR19L/Fas-SM(-), which are defective of SM synthesis and susceptible to methyl-β-cyclodextrin (MβCD)-induced cell death (30Fukasawa M. Nishijima M. Itabe H. Takano T. Hanada K. J. Biol. Chem. 2000; 275: 34028-34034Google Scholar). By an expression cloning method using WR19L/Fas-SM(-) cells and MβCD-based cell selection, we isolated a human cDNA responsible for SM synthase activity. The cDNA clone encodes a peptide of 413 amino acids, named SMS1, which contains a sterile α motif (SAM) domain and four putative transmembrane domains. SMS1 was identical to the peptide that was recently identified as a human SM synthase by Huitema et al. (24Huitema K. van den Dikkenberg J. Brouwers J.F.H.M. Holthuis J.C.M. EMBO J. 2004; 23: 33-44Google Scholar). In serum-free condition, where the cell growth of WR19L/Fas-SM(-) was inhibited, the cells expressing SMS1 cDNA (WR19L/Fas-SMS1) restored the growth ability and accumulation of SM at the surface of the plasma membrane. The restoration of cell growth was also observed when WR19L/Fas-SM(-) cells were maintained in the serum-free medium supplemented with exogenous SM. Here, we show the critical role of SM synthesized through SM synthase in mammalian cell growth, and the localization, active site and biological function of SMS1 are also discussed.EXPERIMENTAL PROCEDURESMaterials—Lysenin, MβCD, and ceramide from bovine brain were purchased from Sigma; PC from egg yolk, SM from bovine brain, and a cell viability assay kit with 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium (WST-8) were from Nacalai tesque (Kyoto, Japan); GP2–293 packaging cell, pLIB retroviral expression vector, and human HeLa cDNA retroviral expression library were from Clontech; d-erythro-C6-NBD-ceramide and C6-NBD-sphingomyelin were from Matreya (Pleasant Gap, PA); l-[U-14C]serine, cytidine 5′-diphospho [methyl-14C]choline, l-3-phosphatidyl [N-methyl-14C]choline, 1,2-dipalmitoyl, and [N-methyl-14C]sphingomyelin were from Amersham Biosciences.Cell Culture—WR19L/Fas cells were kindly gifted from Dr. Yonehara (Institute for Virus Research, Kyoto University). The SM-defective WR19L/Fas-SM(-) cells and the SM-containing WR19L/Fas-SM(+) cells were isolated from the original WR19L/Fas cells by a dilution cloning method. The cells were routinely maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), 50 μm 2-mercaptoethanol, and 75 μg/ml kanamycin in 5% CO2 and 100% humidity at 37 °C. For culture in serum-free medium, the cells were washed, reseeded at 1 × 105 cells/ml, and incubated in the RPMI 1640 medium with 5 μg/ml human insulin and bovine holo transferrin in the presence or absence of 50 μm SM in 5% CO2 at 37 °C. After 48 h incubation, the cell numbers were counted with dye exclusion method using 0.25% trypan blue (Nakalai tesque, Kyoto, Japan).Cell Labeling—The cells were reseeded at 5 × 105 cells/ml in the RPMI 1640 medium with 2% FBS and l-[14C]serine (specific activity; 155 mCi/mmol) and incubated at 37 °C in 5% CO2 for 36 h. The labeled cells were incubated at 37 °C in 5% CO2 for 2 h. The cell lipids were extracted by the method of Bligh and Dyer (19Okazaki T. Bell R.M. Hannun Y.A. J. Biol. Chem. 1989; 264: 19076-19080Google Scholar), applied on a silica Gel 60 TLC plate (Merck), and developed with solvent containing methyl acetate/propanol/chloroform/methanol/0.25% KCl (25:25:25:10:9). The radioactive spots were visualized and quantified by using a BAS 2000 Image Analyzer (Fuji Film).FACS Analyses—The cells were incubated with 500 ng/ml lysenin in the presence of 20 μg/ml propidium iodide (Molecular Probes) at room temperature for 15 min and analyzed with FACS Calibur (BD Biosciences). For detection of SM localized at the plasma membrane, the cells were stained on ice for 30 min with non-toxic lysenin fused to maltose-binding protein (MBP-lysenin) (25Yamaji-Hasegawa A. Makino A. Baba T. Senoh Y. Kimura-Suda H. Sato S.B. Terada N. Ohno S. Kiyokawa E. Umeda M. Kobayashi T. J. Biol. Chem. 2003; 278: 22762-22770Google Scholar), kindly provided by Dr. T. Kobayashi (The Institute of Physical and Chemical Research (RIKEN), Japan). The cells were washed with ice-cold phosphate-buffered saline supplemented with 1% FCS and 0.1% NaN3 and incubated with rabbit anti-MBP antiserum (New England BioLabs, Beverly, MA) on ice for 30 min. After being washed again, the cells were incubated for 30 min with phycoerythrin-conjugated anti-rabbit IgG (Sigma) and subjected to fluorescence-activating cell sorter (FACS) analysis using FACS Calibur. The data analysis was performed by Cell Quest software (BD Biosciences).Confocal Microscopy—For visualization of SM localized at the plasma membrane, the cells settled onto slides coated with poly-l-lysine were fixed in 4% formaldehyde and stained with lysenine-MBP at 4 °C for 45 min followed with anti-MBP. After being stained with a phycoerythrin-conjugated anti-rabbit IgG monoclonal antibody, the cells were examined using confocal microscopy using a Zeiss LSM 310 laser scan confocal microscope (Carl Zeiss, Oberkochen, Germany).Expression Cloning of SMS1 cDNA—The expression cloning method performed in this study was based on the study of Hanada et al. (26Hanada K. Kumagai K. Yasuda S. Miura Y. Kawano M. Fukasawa M. Nishijima M. Nature. 2003; 426: 803-809Google Scholar). Pantropic retroviral particles containing the G glycoprotein of vesicular stomatitis virus (VSV-G) were prepared using a human HeLa cDNA retroviral expression library kit and GP2–293 packaging cells (Clontech). After infection for 24 h, the WR19L/Fas-SM(-) cells were cultured in the RPMI 1640 medium containing 2% FBS overnight. After being washed with serum-free RPMI 1640 medium, the cells were incubated in 1.5 mm MβCD in RPMI 1640 medium for 5 min at 37 °C, replenished with the normal culture medium to a final concentration of FBS at 5%, and then cultured at 37 °C for 60 h. The cells were reseeded, cultured in the RPMI 1640 medium containing 2% FBS overnight, and subjected again to the treatment with appropriate concentrations of MβCD. After a total of two cycles of 1.5 mm MβCD treatment followed by two cycles of 3 mm and two subsequent cycles of 5 mm, an MβCD-resistant variant of WR19L/SM(-) was isolated by a limiting dilution.By genomic PCR using primers specific to the pLIB expression vector (5′ and 3′ pLIB Primer, Clontech), the 2.0-kb cDNA integrated in the genome of the MβCD-resistant cell was amplified and cloned into pGEM-T Easy vector (Promega, Madison, WI). After sequencing and computer analysis, the cDNA was subcloned into the pLIB expression vector and transfected into the WR19L/Fas-SM(-) cells via the VSV-G retroviral particles. A resultant cell was isolated by a limiting dilution method, which was designated WR19L/Fas-SMS1 cells, and subjected to various assays. Integration of the cDNA into the genome of WR19L/Fas-SMS1 cells was confirmed with PCR.Assay for Sphingomyelin Synthase Activity—The cells were homogenized in an ice-cold buffer containing 20 mm Tris-HCl, pH 7.4, 2 mm EDTA, 10 mm EGTA, 1 mm phenylmethylsulfonyl fluoride, and 2.5 μg/ml leupeptin. The lysates containing 500 μg of cell protein were added to a reaction solution containing 10 mm Tris-HCl, pH 7.5, 1 mm EDTA, 20 μm C6-NBD-ceramide, 120 μm PC and incubated at 37 °C for 30 min. The lipids were extracted by the method of Bligh and Dyer (19Okazaki T. Bell R.M. Hannun Y.A. J. Biol. Chem. 1989; 264: 19076-19080Google Scholar), applied on the TLC plates, and developed with solvent containing chloroform/methanol/12 mm MgCl2 in H2O (65:25:4). The fluorescent lipids were visualized by FluorImager SI system (Amersham Biosciences). For the assay for transferase activity, 20 μm ceramide and 120 μm [N-methyl-14C]PC (specific activity; 57 mCi/mmol) or [methyl-14C]CDP-choline (specific activity; 54 mCi/mmol) were used in the reaction solution instead of the NBD-ceramide and PC. The radioactive spots were visualized using the BAS 2000 system.Assay for Viability and Growth Rate of Cells Exposed to MβCD and Lysenin—For the assay using MβCD, 1 × 106 of the cells were washed and resuspended in 1 ml of the serum-free RPMI 1640 medium, treated with appropriate concentrations of MβCD, and incubated in 5% CO2 at 37 °C for 5 min. After the addition of 1 ml of the normal culture medium, the cells were further incubated for 12 h. The viability of the cells was measured using a cell viability kit with WST-8 (Nakalai tesque). For the assay using lysenin, 7 × 105 of the cells were washed and resuspended in 1 ml of prewarmed phosphate-buffered saline, treated with the appropriate concentrations of lysenin and incubated in 5% CO2 at 37 °C for 1 h. After the addition of FBS, the cell number was counted with the 0.25% trypan blue dye exclusion method.RESULTS AND DISCUSSIONMouse Lymphoid Cells Defective of Sphingomyelin Synthase Activity—During investigation of the sphingolipid metabolism in mouse lymphoid cells named WR19L/Fas, which overexpress the human Fas antigen, the variant clones altering SM synthase activity (from 150 to nearly 0 pmol/mg protein/h) have been isolated. One of the variants (clone 6) severely diminished the SM synthase activity (Fig. 1A). Conversion of C6-NBD-ceramide to C6-NBD-SM in the cell lysate of the clone 6, named WR19L/Fas-SM(-), was not detected on a TLC plate, in contrast to the clone 2 showing the highest SM synthase activity, named WR19L/Fas-SM(+) (Fig. 1B). This finding was supported by the fact that WR19L/Fas-SM(-) cells did not synthesize [14C]serine-labeled SM (Fig. 1C).Lysenin is reported as an SM-direct cytolysin purified from the earthworm (27Yamaji A. Sekizawa Y. Emoto K. Sakuraba H. Inoue K. Kobayashi H. Umeda M. J. Biol. Chem. 1998; 273: 5300-5306Google Scholar), for which binding to SM causes poring of the plasma membrane and subsequent cell death (22Hanada K. Hara T. Fukasawa M. Yamaji A. Umeda M. Nishijima M. J. Biol. Chem. 1998; 273: 33787-33794Google Scholar, 23Shakor A.B. Czurylo E.A. Sobota A. FEBS Lett. 2003; 542: 1-6Google Scholar, 25Yamaji-Hasegawa A. Makino A. Baba T. Senoh Y. Kimura-Suda H. Sato S.B. Terada N. Ohno S. Kiyokawa E. Umeda M. Kobayashi T. J. Biol. Chem. 2003; 278: 22762-22770Google Scholar). Hanada et al. (22Hanada K. Hara T. Fukasawa M. Yamaji A. Umeda M. Nishijima M. J. Biol. Chem. 1998; 273: 33787-33794Google Scholar) previously showed that Chinese hamster ovary cells, which express SM in the outer surface of the plasma membrane, were sensitive to lysenin-induced cell death. They also showed that reduced accumulation of SM in the variant Chinese hamster ovary cells, LY-A and LY-B, causes the significant resistance against lysenin (22Hanada K. Hara T. Fukasawa M. Yamaji A. Umeda M. Nishijima M. J. Biol. Chem. 1998; 273: 33787-33794Google Scholar). In LY-A cells, the reduction of SM is caused by the lack of non-vesicular transporter for ceramide between endoplasmic reticulum (ER) and Golgi apparatus (CERT) (26Hanada K. Kumagai K. Yasuda S. Miura Y. Kawano M. Fukasawa M. Nishijima M. Nature. 2003; 426: 803-809Google Scholar), whereas in LY-B cells, it is due to the lack of LCB1, a component of serine palmitoyltransferase (22Hanada K. Hara T. Fukasawa M. Yamaji A. Umeda M. Nishijima M. J. Biol. Chem. 1998; 273: 33787-33794Google Scholar). CERT is involved in SM synthesis by transferring ceramide from the endoplasmic reticulum to the cytoplasmic surface of Golgi apparatus (26Hanada K. Kumagai K. Yasuda S. Miura Y. Kawano M. Fukasawa M. Nishijima M. Nature. 2003; 426: 803-809Google Scholar, 31Fukasawa M. Nishijima M. Hanada K. J. Cell Biol. 1999; 144: 673-685Google Scholar). Recently, Kobayashi and co-workers (25Yamaji-Hasegawa A. Makino A. Baba T. Senoh Y. Kimura-Suda H. Sato S.B. Terada N. Ohno S. Kiyokawa E. Umeda M. Kobayashi T. J. Biol. Chem. 2003; 278: 22762-22770Google Scholar) reported a modified lysenin, which specifically binds to SM without the induction of cell death. By using the modified lysenin conjugated with MBP, we examined the accumulation of SM on the cellular surface of WR19L/Fas-SM(-) cells. Binding of the modified lysenin was positively detected in WR19L/Fas-SM(+) cells but not in WR19L/Fas-SM(-) cells by FACS analysis and confocal microscopy using anti-MBP antibody (Fig. 1D), indicating that the accumulation of SM on the outer surface of the WR19L/Fas-SM(-) cells was severely reduced. The results were supported by the fact that WR19L/Fas-SM(+) cells underwent cell death by treatment with the cytotoxic lysenin, whereas WR19L/Fas-SM(-) cells did not, when we examined cell viability by staining with propidium iodide and subsequent FACS analysis (Fig. 1E). These facts suggest that the severe reduction of SM at the cellular surface of WR19L/Fas-SM(-) cells is due to the lack of enzymatic activity of SM synthase.Expression Cloning of a Human cDNA Responsible for Resistance to Methyl-β-cyclodextrin-induced Cell Death—It has been reported that SM strongly interacts with cholesterol in biological and artificial membranes (28Slotte J.P. Chem. Phys. Lipids. 1999; 102: 13-27Google Scholar) and that SM is required to form the membrane microdomains (lipid rafts) related to cell functions such as cell death and growth (29Ostermeyer A.G. Beckrich B.T. Ivarson K.A. Grove K.E. Brown D.A. J. Biol. Chem. 1999; 274: 34459-34466Google Scholar, 30Fukasawa M. Nishijima M. Itabe H. Takano T. Hanada K. J. Biol. Chem. 2000; 275: 34028-34034Google Scholar). LY-A cells were sensitive to MβCD-induced cell death due to the decrease of the SM level in plasma membrane (30Fukasawa M. Nishijima M. Itabe H. Takano T. Hanada K. J. Biol. Chem. 2000; 275: 34028-34034Google Scholar). We similarly observed that WR19L/Fas-SM(-) cells were highly sensitive to MβCD-induced cell death, whereas WR19L/Fas-SM(+) cells were not (Fig. 2D). This finding allowed us to screen WR19L/Fas-SM(-) cells complemented with the ability of SM synthesis using MβCD as a selective agent. Using a pantropic retroviral transfection system, WR19L/SM(-) cells were transfected with a cDNA expression library of the human HeLa cell, and the variant cells, which were resistant to MβCD due to the expression of SM in the plasma membrane, were selected. The variant cells were isolated to a single clone by a limiting dilution method. The purified cells integrated a human cDNA of 1967 bp in the genome, which encodes a peptide of 413 amino acids with 48.6 kDa of a predicted molecular mass (Fig. 2A). The BLAST algorithm (32Altschul S.F. Gish W. Miller W. Myers E.W. Lipman D.J. J. Mol. Biol. 1990; 215: 403-410Google Scholar) and the SOSUI program (33Hirokawa T. Boon-Chieng S. Mitaku S. Bioinformatics (Oxf.). 1998; 14: 378-379Google Scholar) suggested that this peptide carries a SAM domain in the N-terminal region and four transmembrane helices, respectively (Fig. 2B). The SAM domain is suggested to be involved in signal transduction, development, and transcriptional regulation (34Bork P. Koonin E.V. Nat. Genet. 1998; 18: 313-318Google Scholar, 35Barrera F.N. Poveda J.A. Gonzalez-Ros J.M. Neira J.L. J. Biol. Chem. 2003; 278: 46878-46885Google Scholar). A variety of proteins such as ephrin-related receptor tyrosine kinase, a variant of p53 (p73), and DAG kinase δ contain the SAM domain(s), which may play a role in protein-protein or protein-lipid interaction (35Barrera F.N. Poveda J.A. Gonzalez-Ros J.M. Neira J.L. J. Biol. Chem. 2003; 278: 46878-46885Google Scholar, 36Schultz J. Ponting C.P. Hofmann K. Bork P. Protein Sci. 1997; 6: 249-253Google Scholar). Recently, Huitema et al. (24Huitema K. van den Dikkenberg J. Brouwers J.F.H.M. Holthuis J.C.M. EMBO J. 2004; 23: 33-44Google Scholar) reported a family of SM synthases using a bioinformatics and functional cloning strategy in yeast. They identified the human cDNAs encoding the peptides that shared a sequence motif with the lipid phosphate phosphatases and Aur1p proteins required for inositolphosphorylceramide production in yeast (24Huitema K. van den Dikkenberg J. Brouwers J.F.H.M. Holthuis J.C.M. EMBO J. 2004; 23: 33-44Google Scholar). One of the human peptides, SMS1, was identical to our peptide. They further demonstrated that SMS1 was localized at Golgi apparatus and predicted the six transmembrane domains and an exoplasmic catalytic site, which is consistent with the characteristics of SM synthase suggested previously (37Futerman A.H. Stieger B. Hubbard A.L. Pagano R.E. J. Biol. Chem. 1990; 265: 8650-8657Google Scholar, 38van Helvoort A. Stoorvogel W. van Meer G. Burger N.J. J. Cell Sci. 1997; 110: 781-788Google Scholar, 39Elmendorf H.G. Haldar K. J. Cell Biol. 1994; 124: 449-462Google Scholar). Molecular structure of SMS1, including the transmembrane domains, should be clarified by further detailed analysis.Fig. 2Expression cloning of a human cDNA responsible for cellular resistance to methyl-β-cyclodextrin. A, nucleotide sequence and predicted amino acid sequence of SMS1. Putative SAM domain sequence and transmembrane (TM) regions are indicated with the thin and thick underline, respectively. SAM domain and transmembrane domains were predicted by the BLAST algorithm and SOSUI program, respectively. B, hydropathy plot of the amino acid sequence of SMS1 analyzed by the method of Kyte and Doolittle (45Kyte J. Doolittle R.F. J. Mol. Biol. 1982; 157: 105-132Google Scholar). Positions of the putative SAM domain and transmembrane regions are indicated with horizontal bars. C, integration of SMS1 cDNA (2 kb) into the genome of WR19L/Fas-SM(-) and -SMS1 cells was examined by PCR. D, the viability of WR19L/Fas-SM(-), -SM(+), and -SMS1 cells exposed to various concentrations of MβCD. The viability of the cells was examined using WST-8. The data were the average and 1 S.D. obtained from three independent experiments.View Large Image Figure ViewerDownload (PPT)It was recently proposed by Luberto et al. (40Luberto C. Stonehouse M.J. Collins E.A. Marchesini N. El-Bawab S. Vasil A.I. Vasil M.L. Hannun Y.A. J. Biol. Chem. 2003; 278: 32733-32743Google Scholar) that the Pseudomonas PlcH gene product, which is a secreted protein, is a putative SM synthase. The SMS1 peptide was suggested to be an integral membrane protein and did not show any significant homology with the PlcH product.The SMS1 cDNA Is Responsible for Sphingomyelin Synthase in Mammalian Cells—Huitema et al. (24Huitema K. van den Dikkenberg J. Brouwers J.F.H.M. Holthuis J.C.M. EMBO J. 2004; 23: 33-44Google Scholar) demonstrated the SM synthase activity in the yeast cells expressing SMS1 cDNA. Here, we demonstrated that the loss of SM synthesis in the SM-defective mammalian cells was complemented with SMS1 cDNA. WR19L/Fas-SM(-) cells transfected with SMS1 cDNA, named WR19L/Fas-SMS1 (Fig. 2C), restore the resistance against MβCD-induced cell death (Fig. 2D). Radiolabeling of cellular lipids with [14C]serine revealed that [14C]SM synthesis was also restored in WR19L/Fas-SMS1 cells (Fig. 3A), and whole cell lysate from WR19L/Fas-SMS1 cells generated C6-NBD-SM in the presence of C6-NBD-ceramide and PC (Fig. 3B). These results strongly suggest that the SMS1 cDNA is indispensable for SM synthase activity in mammalian cells. Furthermore, SM synthase activity in WR19L/Fas-SMS1 cells was detected in the presence of [14C]PC but not [14C]CDP-choline (Fig. 3C), suggesting that PC was a phosphocholine donor for SM synthesis by SMS1. These results indicate that the SMS1 protein possesses the characteristics consistent with those of SM synthase reported elsewhere previously (8Voelker D.R. Kennedy E.P. Biochemistry. 1982; 21: 2753-2759Google Scholar).Fig. 3Restoration of SM synthesis and SM synthase activity in WR19L/Fas-SMS1. A, the cellular lipids of WR19L/Fas-SM(-) and -SMS1 labeled with [14C]serine were assessed by TLC as described in Fig. 1. Cer, ceramide; GC, glucosylceramide; PE, phosphatidylethanolamine; PS, phosphatidylserine. B, SM synthase activity of WR19L/Fas-SM(-) and -SMS1 cells was assessed in the absence of UDP-glucose as described in the legend for Fig. 1. C, SM synthase activity was assessed using the radiolabeled PC and CDP-choline as the donor of phosphocholine moiety. The radiolabeled products developed on the TLC plate were visualized by BAS 2000 system. The data were the representative of three independent experiments.View Large Image Figure ViewerDownload (PPT)The SMS1 cDNA Is Essential for Growth in Mammalian Cells—In contrast to the role of ceramide in cell death, the biological implications of SM are still ambiguous.
Abstract Although exposure-directed busulfan (BU) dosing can improve allogeneic hematopoietic stem cell transplantation outcomes, there is still large variability in BU exposure with test-dose alone due to BU clearance change caused by drug interactions. We conducted a single-arm Phase II trial (UMIN000014077) using the combined test-dose and therapeutic drug monitoring strategy (PK-guided group) and compared the outcomes with an historical cohort receiving a fixed-dose (fixed-dose group). The first eight and second eight doses were adjusted based on the area under the blood concentration-time curve (AUC) of the test and first doses, respectively. The BU clearance at the first dose decreased in more patients receiving fludarabine (FLU) than those receiving BU and cyclophosphamide (BU/CY) from the test dose. The simulated total AUC with test-dose only was significantly higher in patients receiving FLU than in those receiving BU/CY, but the simulated AUC with the combined strategy was comparable. The 100-day progression-free survival in PK-guided group was not inferior to that in fixed-dose group. For the FLU-containing regimens, the PK-guided group showed decreased relapse, and favorable overall survival at one year. The combined strategy effectively controlled the BU exposure close to the target levels, potentially improving efficacy, especially in patients receiving the FLU-containing regimen. Clinical Trial Registry #UMIN000014077
17066 Background: The information generated in daily practices is critical to assure safety and efficacy of therapeutic interventions. Clinical investigators are faced with enormous amounts of data and a greater need to organize it in a meaningful and coherent manner than ever before. Computerization could offer many advantages that clinical data systematically accumulated in the course of routine medical care can provide researchers with the clues to resolve many medical questions. Methods: We developed a novel clinical database system, named CyberOncology, integrated in EMR of Kyoto University Hospital. It contained summarized treatment history, national cancer registry and consecutive clinical database. All adverse events according CTCAE ver.3, response to the treatment based on RECIST criteria and survival data were collected. The medical staffs have routinely used the CyberOncology since its starting on October 2003. The CyberOncology directly collected all data concerning oncology management of inpatient and outpatient care from EMR, and simultaneously analyzed clinical outcomes. Results: For three years since October 2003, consecutive 1,516 new cancer patients including 590 GI, 435 lung, 234 breast cancer, and 140 lymphoma with 19,767 chemotherapeutic administrations were registered in the CyberOncology. Main benefits are improvement of the quality of patient care and safety, practice standardization, and the quickness and the reliability of collecting the data. Moreover, it had the capability to serve a cross-sectional approach of cancer and drug-orientated analysis. For example, this system can easily provided with a review of clinical practice in a real time manner. Conclusions: The CyberOncology form integrated EMR successfully meets requirements of electronic case report. It has been useful in monitoring outcomes of care, effectiveness, efficiency and adherence to clinical trial. No significant financial relationships to disclose.
