Alkaline sphingomyelinase (alk-SMase) hydrolyzes dietary sphingomyelin and generates sphingolipid messengers in the gut. In the present study, we purified the enzyme, identified a part of the amino acid sequence, and found a cDNA in the GenBank TM coding for the protein. The cDNA contains 1841 bp, and the open reading frame encodes 458 amino acids. Transient expression of the cDNA linked to a Myc tag in COS-7 cells increased alk-SMase activity in the cell extract by 689-fold and in the medium by 27-fold. High activity was also identified in the anti-Myc immunoprecipitated proteins and the proteins cross-reacted with anti-human alkSMase. Northern blotting of human intestinal tissues found high levels of alk-SMase mRNA in the intestine and liver. The amino acid sequence shared no similarity with acid and neutral SMases but was related to the ecto-nucleotide phosphodiesterase (NPP) family with 30 -36% identity to human NPPs. Alk-SMase has a predicted signal peptide domain at the N terminus and a signal anchor domain at the C terminus. The ion-binding sites and the catalytic residue of NPPs were conserved, but the substrate specificity domain was modified. Alk-SMase had no detectable nucleotidase activity, but its activity against sphingomyelin could be inhibited by orthovanadate, imidazole, and ATP. In contrast to NPPs, alk-SMase activity was not stimulated by divalent metal ions but inhibited by Zn 2؉ . Differing from NPP2, the alk-SMase cleaved phosphocholine but not choline from lysophosphatidylcholine. Phylogenetic tree indicated that the enzyme is a new branch derived from the NPP family. Two cDNA sequences of mouse and rat that shared 83% identity to human alk-SMase were identified in the GenBank TM . In conclusion, we identified the amino acid and cDNA sequences of human intestinal alk-SMase, and found that it is a novel ectoenzyme related to the NPP family with specific features essential for its SMase activity. Sphingomyelin (SM)1 is a component of all mammalian cell membranes particularly the plasma membrane and the lysosomal membrane. SM is also a dietary component and is mainly present in milk, eggs, meat, and marine products (1, 2). Hydrolysis of SM generates ceramide, sphingosine, and sphingosine 1-phosphate that have regulatory effects on numerous cellular functions such as proliferation, differentiation, and apoptosis (3, 4). At least five types of sphingomyelinase (SMase) have been identified, of which acid and neutral SMases have been cloned (5-9). An enzyme that catalyzes hydrolysis of SM with optimal alkaline pH was first identified in the intestinal content of human and intestinal mucosa of rat and pig by Nilsson (10) and was named alkaline SMase (alkSMase) thereafter (11). Previous studies indicated that alkSMase may be responsible for digestion of dietary SM and for hydrolysis of endogenous SM derived from bile and from the brush borders of sloughed mucosal cells.SM metabolism in the intestine may have implications in colon cancer development. Dietary supplement with SM and ceramide analogues...

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p21/waf1/cip1 and mdm‐2 are downstream effectors of p53. p21 plays a major role in negatively regulating cell‐cycle progression, while mdm‐2 inhibits p53 effects, and its role has been implicated in oncogenesis. In this study, we investigated the expression profiles of p21, mdm‐2 and p53 in human breast‐carcinoma tissues. The aim was to determine whether a correlation exists between the expression profiles of these markers and tumor differentiation, ER status and prognosis. We studied tumor specimens obtained from 106 patients and found a highly significant association among low histology grade, p53 over‐expression, high mdm‐2 expression and lack of p21 expression. Our studies also demonstrate that, in human breast cancer, low levels of p21 and higher mdm‐2 levels directly correlate with the onset of lymph‐node metastases and shortened patient survival. Furthermore, the expression profiles of p21, mdm‐2 and p53 were independently correlated with patient survival. Int. J. Cancer 74:529–534, 1997. © 1997 Wiley‐Liss, Inc.

Tumor angiogenesis is a critical element of cancer progression, and strategies for its selective blockade are still sought. Here, we examine the angiogenic effects of Globo-H ceramide (GHCer), the most prevalent glycolipid in a majority of epithelial cancers and one that acts as an immune checkpoint. Here, we report that GHCer becomes incorporated into endothelial cells through the absorption of microvesicles shed from tumor cells. In endothelial cells, GHCer addition induces migration, tube formation, and intracellular Ca 2þ mobilization in vitro and angiogenesis in vivo. Breast cancer cells expressing high levels of GHCer displayed relatively greater tumorigenicity and angiogenesis compared with cells expressing low levels of Globo-H. Clincally, GHCer þ breast cancer specimens contained higher vessel density than GHCer À breast cancer specimens. Mechanistic investigations linked the angiogenic effects of GHCer to its endocytosis and binding to TRAX, with consequent release of PLCb1 from TRAX to trigger Ca 2þ mobilization. Together, our findings highlight the importance of GHC as a target for cancer therapy by providing new information on its key role in tumor angiogenesis. Cancer Res; 74(23); 6856-66. Ó2014 AACR.

IntroductionMer and Tyro3 are receptor tyrosine kinases important for the phagocytosis of apoptotic cells. Together with Axl, they constitute the TAM receptor family. These receptors can be shed from the cell membrane and their soluble extracellular regions can be found in plasma. The objective of this study was to elucidate whether the plasma levels of soluble Mer (sMer) and Tyro3 (sTyro3) were increased in systemic lupus erythematosis (SLE), rheumatoid arthritis (RA), or critical limb ischemia (CLI).MethodsELISA kits were used to test plasma concentrations in controls and in patients with SLE, RA or CLI.ResultsIncreased levels of, in particular, sMer and, to some extent, sTyro3, were found in patients with SLE or RA, but not in patients with CLI. Patients with SLE demonstrated the highest sMer levels and there was a strong correlation to higher SLE disease activity score (SLEDAI). In contrast, in patients with RA, the sMer levels did not correlate with the disease activity score (DAS). In SLE, sMer levels were particularly high in those with lupus nephritis, patients who also had decreased C1q levels and increased titers of anti-DNA antibodies. After therapy, the plasma concentrations of sMer decreased in parallel to the decrease in SLEDAI score.ConclusionsThe plasma concentrations of sMer and sTyro3 were significantly increased in patients with active SLE and RA, suggesting the TAM receptor shedding was affected by these autoimmune diseases. In particular, sMer was increased in SLE, the plasma levels of sMer reflecting disease activity.