Expression levels of GM3 synthase is transcriptionally regulated in HL60 cells differentiated in monocytoid lineage by phorbol esters

INTRODUCTION: During bi-directional differentiation of human myelogenous leukemia cell line HL-60 into monocytoid and granulocytoid lineages, ganglioside GM3 and neolacto series gangliosides (NeuAc-nLCs) are expressed in differentiation direction-specific manner (1). That is, GM3 markedly increases during monocytic differentiation of HL-60 cells induced by 12-O-tetradecanoyilphorbol-13-acetate (TPA), whereas NeuAc-nLCs noticeably increase in granulocytic differentiation induced by all-trans retinoic acid (RA). These observations suggest that the accumulation of specific gangliosides on the cell membrane plays an important role as a trigger in differentiation induction and as determinant of the differentiation direction in human hematopoietic cell lines (1). It is known that two key upstream glycosyltransferases, Lc3Cer synthase and GM3 synthase, play a critical role regulating the glycosphingolipid biosynthesis in HL-60 cells during bi-directional differentiation (2), but the mechanisms controlling expression and activity levels of these enzymes have not yet been elucidated. In this study our attention is directed to investigate the regulation of GM3 synthase activity. MATERIALS AND METHODS: HL-60 cells were grown in RPMI 1640 complete medium at 37\ub0C in a humidified atmosphere enriched with 5% CO2. Granulocytoid differentiation of HL-60 cells was induced by treatment with 1 mM RA for 48 hours; macrophage-like cell differentiation was produced by 4 nM TPA addition to the culture medium for the same period of time. Acidic and neutral glycolipid profiles of control, RA- and TPA-treated cells were quali-quantitatively analysed by HP-TLC and digital scanning of the plates. GM3 synthase activities were determined in control, RA- and TPA-treated cells by an in vitro radioactive assay using 50 mg and 100 mg of the microsomal enriched protein fraction as enzyme source. mRNA expression levels of GM3 synthase gene was determined by RT-PCR. The house-keeping gene encoding for hypoxantine phospho-ribosyl transferase (HPRT) was used as internal standard for quantitative evaluation of the RT-PCR products. RESULTS: After 48 hours RA treatment, a 30% granulocytic differentiation degree of HL-60 cells was evaluated by conventional cytoplasmic/nuclear histochemical staining of the cells. On the contrary, quite 80% of TPA-treated cells showed evident macrophage-like adherent ability and prominent pseudopods, phenotipic markers of differentiation. Indeed, no modification in the glycosphingolipid profiles, in the enzyme activities and in mRNA expression levels of the crucial glycosyltransferases (Lc3Cer synthase and GM3 synthase) were observed in RA-treated cells. On the other hand, in TPA-treated cells there is a sensible increase in ganglioside GM3 content, accompanied by a consistent up-regulation of GM3 synthase activity with respect to undifferentiated and to RA-treated cells. Through quantitative RT-PCR experiments performed on total RNA from undifferentiated, RA- and TPA-treated HL-60 cells, we demonstrate the strict correlation between GM3 synthase activity and its mRNA level: the GM3 synthase transcript is present in equal amount in either undifferentiated and RA-treated cells, but it is dramatically increased (quite 3 times) in TPA-treated cells. These results first give support to a regulation mechanism at the transcriptional level for this enzyme. REFERENCES (1) H. Nojiri et al., Characteristic expression of glycosphingolipid profiles in the bipotential cell differentiation of human promyelocytic leukemia cell line HL-60. Blood 64, 2:534-541 (1984); (2) M. Nakamura et al., Total metabolic flow of glycosphingolipid biosynthesis is regulated by UDP-GlcNAc:Lactosylceramide beta-1,3-N-Acetylglucosaminyltransferase and CMP-NeuAc:Lactosylceramide alfa-2,3sialyltransferase in human hematopoietic cell line HL-60 during differentiation. J. Biol. Chem. 267, 33: 23507-23541 (1992)

ISOLATION AND CHARACTERIZATION OF A NOVEL ST3Gal V mRNA ISOFORM FROM HUMAN PLACENTA

INTRODUCTION: ST3Gal V (EC 2.4.99.9, GM3 synthase) is a key enzyme in the biosynthesis of gangliosides, a large and heterogeneous family of sialic acid-containing glycosphingolipids that, as mediator of cell-cell interactions and modulators of signalling transduction, play fundamental roles in many both physiological and pathological cellular processes (i.e., proliferation, differentiation, oncogenesis) (1). It catalyses the transfer of a sialic acid moiety from CMP-sialic acid onto LacCer producing GM3, a ganglioside ubiquitously distributed on the plasma membrane of all the eukaryotic cells, representing the common precursor of almost all ganglio-series gangliosides (2). However, the homologous rat protein purified from brain and liver can use other glycolipids as acceptor substrates, including GalCer, GlcCer, aGM2, and aGM1, although with a lower extent of specificity respect to LacCer (3). Contrasting results are also reported about the intracellular localization of the protein: GM3 synthase activity has been detected in both proximal and distal compartments of Golgi apparatus (4). Human ST3Gal V results expressed in a tissue-specific manner, especially in brain, muscle, testis, and placenta, and only one transcript, of about 2.4 kb, has been detected in all the tissues (5). Human ST3Gal V cDNAs have been already cloned from both TPA-differentiated HL60 cells and fetal and adult brain, and several mRNA variants have been identified (5). They differ in the 5\u2019-UTR sequences, but all of them seem to contain an identical coding region; the substrate activity of the encoded protein (362 aminoacids with a predicted molecular mass of 41.7 kDa) is highly restricted to LacCer. Studies on the structural organization and transcriptional regulation of the gene (6) also provided contrasting, but stimulating, results. MATERIALS AND METHODS: The complete ST3Gal V cDNA from human placenta was obtained by the 5\u2019- and 3\u2019-RACE technology (SMART RACE cDNA Amplification Kit, Clontech) using total RNA as template. The sequence of the PCR product was determined by M-Medical\u2019s DNA sequencing service. The transcription initiation site was evaluated by primer extension analysis. The translation initiation site was identified by in vitro experiments, using the TNT\uae T7 Quick coupled transcription/translation system (Promega), and it was confirmed by in vivo analyses. The isolated cDNA protein product, expressed as fusion protein with EGFP in CV1 cells using pEGFP-N3 as expression vector, was detected by western blot with specific anti-GFP antibodies. The enzymatic activity of the isolated cDNA protein product was assayed in rat mammary adenocarcinoma cells stably transfected with the isolated cDNA using the pRC/CMV (Promega) as expression vector; the enzymatic activity was determined by an in vitro radioactive assay using the microsomal enriched protein fraction as enzyme source, CMP-[14C]sialic acid as donor substrate, and different glycolipids as acceptor substrates. The genomic structure of the human ST3Gal V gene has been determined through a human genome BLAST homology search of the public database (GenBank) using the isolated cDNA as query sequence. RESULTS: A cDNA, consisting of 2149 bp (the poli-A tail is lacking) and showing high sequence identity with all the human ST3Gal V cDNAs until now registered in GeneBank, has been successfully isolated and cloned from human placenta. Primer extension experiments confirmed the transcription initiation site. Not surprisingly, it differs from all the other human cDNAs in the 5\u2019-end sequence, but, with respect to the other ones, it contains a new, possibly translation initiation, ATG codon; it is located upstream and in frame with the ATG indicated as translation initiation site in all the other human ST3Gal V cDNAs. In vitro and in vivo analyses on the placental cDNA product showed that it predominantly produces a protein with a larger molecular mass (about 44 kDa), having a higher substrate specifity for LacCer; however, GalCer, aGM1, and aGM2 could serve as substrates, although to a much lesser extent. Finally, a human genome BLAST homology search in the public database, using the isolated human placental cDNA as the query sequence, showed that the gene consists of seven exons which span over 28.5 kb, with exons ranging in size up to 1242 bp. This study, together with previously reported ones (3,5,6), suggests that the human ST3Gal V gene specifies at least two isozymes, having a tissue-specific expression, a different amino-terminal sequence, and a different substrate specificity (and why not different intracellular localization?). 1) Hakomori S.I. (2000) Glycoconj. J. 17, 143; Hakomori S.I. (2000) Glycoconj. J. 17, 627. 2) Kolter T. et al. (2002) J. Biol. Chem. 277, 25859. 3) Preuss U. et al. (1993) J. Biol. Chem. 268, 26273; Melkerson-Watson L.J. et al. (1991) J. Biol. Chem. 266, 4448. 4) Maccioni H.J.F. et al. (1999) Biochim. Biophys. Acta 1437, 101 5) Ishii A. et al. (1998) J. Biol. Chem. 273, 31652; Kapitonov D. et al.(1999) Glycoconj J. 16, 337. 6) Kim K.W. et al. (2001) Gene 273, 163; Zeng G. et al. (2003) Biochim. Biophys. Acta 1625, 30

Intramolecular Pd(II)-Catalyzed Cyclization of Propargylamides: Straightforward Synthesis of 5-Oxazole-carbaldehydes

(Chemical Equation Presented) Direct synthesis of 2-substituted 5-oxazolecarbaldehydes was performed by intramolecular reaction of propargylamides through treatment with a catalytic amount of Pd(II) salts in the presence of a stoichiometric amount of reoxidant agent. The heterocyclization process was well-tolerated by a wide range of aryl, heteroaryl, and alkyl propargylamides. This protocol constitutes a valuable synthetic pathway to 5-oxazolecarbaldehydes, alternative to the formylation on oxazole rings, often unsatisfactory in term of regioselectivity and yields

CLONING OF THE GM3 SYNTHASE cDNA FROM HUMAN PLACENTA AND GENOMIC ORGANISATION OF THE GENE

INTRODUCTION: Gangliosides are a large family of sialic acid-containing glycosphingolipids that play important roles in a large variety of biological processes. Both their functions and their biosynthetic pathways are currently clarified (1, 2). In vertebrates, almost all the ganglio-series gangliosides are synthesized from a common precursor, ganglioside GM3, which has the simplest structure among the major ganglioside. GM3 itself is known to participate in induction of differentiation, modulation of proliferation, signal transduction and integrin-mediated cell adhesion. GM3 synthase (EC 2.4.99.9, ST3Gal V) is the enzyme involved in the last step of GM3 biosynthesis: it catalyses the transfer of a sialic acid moiety from CMP-sialic acid onto lactosylceramide, forming an a2-3 linkage. Whereas GM3 is ubiquitously distributed in the plasma membranes of all eukaryotic cells, GM3 synthase results expressed in a tissue-specific manner, especially in brain, placenta, muscle and testis (3). Although its cDNA has been cloned from some mouse (4, 5) and human tissues (3, 6), studies on the genomic structure (7, 8) and on its transcriptional regulation (8, 9) provides contrasting results. MATERIALS AND METHODS: To isolate the complete coding sequence of the gene of GM3 synthase from human placenta we used the 5\u2019- and 3\u2019-Rapid Amplification of cDNA Ends technology (SMART RACE cDNA Amplification Kit, Clontech) using, as specific primers, oligonucleotides derived from the human GM3 synthase cDNA sequence from differentiated HL60 cells (3). The identity of some amplified DNA fragments was confirmed by Southern blot analysis (Gene ImagesTM AlkPhos DirectTM labelling and detection system, Amersham Pharmacia Biotech). The different PCR products were cloned into the pCR2.1 vector (TA Cloning Kit, InVitrogen) and the nucleotide sequence was determined (\u201cProgetto Camilla\u201d, M-Medical). The genomic structure of the human GM3 synthase gene has been determined through a human genome BLAST homology search of the public database (GenBank) using the GM3 synthase cDNA from human placenta as the query sequence. RESULTS: A cDNA, consisting of 2149 bp and showing high sequence homology with those encoding the human GM3 synthase from other human tissues (3, 6), has been successfully isolated and cloned from human placenta. Notwithstanding our approach, our cDNA has not the poli(A) tail. Between our cDNA and the other published ones, the major difference is in the 5\u2019-end, according to the existence of different promoter regions, responsible for tissue-specific expression of the gene. Furthermore, the cDNA from the human placenta contains, upstream and in frame with the ATG indicated as translation initiation site for the GM3 synthase of HL60 cells, another ATG codon inserted in a sequence compatible with Kozak\u2019s rule, suggesting that the protein of the human placenta could have an additional portion in NH2-terminus. The complete and partial coding regions of the human placenta cDNA are going to be cloned in an expression vector, under the control of the CMV promoter, in order to evaluate their GM3 synthase activity. The results of the human genome BLAST homology search of the public database using the GM3 synthase cDNA from human placenta as the query sequence showed that the gene consists of seven exons which span over 28.5 kb, with exons ranging in size up to 1242 bp. All exon-intron boundaries follows the GT-AG rule (10). 1) Hakomori S.I. (2000) Glycoconj. J. 17, 627-647 2) Kolter T. et al. (2002) J. Biol. Chem. 277, 25859-25862 3) Ishii A. et al. (1998) J. Biol. Chem. 273, 31652-31655 4) Kono M. et al. (1998) Biochem. Biophys. Res. Comm. 253, 170-175 5) Fukumoto S. et al. (1999) J. Biol. Chem. 274, 9271-9276 6) Kapitonov D. et al.(1999) Glycoconj J. 16, 337-350 7) Kim K.W. et al. (2001) Gene 273, 163-171 8) Kim S.W. et al. (2002) ) Biochim. Biophys. Acta 1578, 84-89 9) Zeng G. et al. (2003) Biochim. Biophys. Acta 1625, 30-35 10) Breathnach R. and Chambon P. (1981) Annu. Rev. Biochem. 50, 349-38

ISOLATION AND CHARACTERIZATION OF THE GM3 SYNTHASE cDNA FROM HUMAN PLACENTA

It is known that gangliosides have various important biological functions, and their functions as well as their biosynthesis are currently clarified (1, 2). In vertebrates, almost all the ganglio-series gangliosides are synthesized from a common precursor, ganglioside GM3, which has the simplest structure among the major gangliosides. GM3 itself is known to participate in induction of differentiation, modulation of proliferation, signal transduction and integrin-mediated cell adhesion. GM3 synthase (EC 2.4.99.9, ST3Gal V) is the enzyme involved in the last step of GM3 biosynthesis: it catalyses the transfer of a sialic acid moiety from CMP-sialic acid onto lactosylceramide, forming an a2-3 linkage. Whereas GM3 is ubiquitously distributed in the plasma membranes of all eukaryotic cells, GM3 synthase results expressed in a tissue specific manner, especially in brain, placenta, muscle and testis (3). Many important issues, such as human cDNA identification and characterization, genomic structure and regulation of gene expression, are still open. To isolate the coding sequence of the gene of GM3 synthase from human placenta we used the 5\u2019- and 3\u2019-Rapid Amplification of cDNA Ends technology (SMART RACE cDNA Amplification Kit, Clontech) using, as specific primers, oligonucleotides derived from the human GM3 synthase cDNA sequence from differentiated HL60 cells (3). The different PCR products were cloned into the pCR2.1 vector (TA Cloning Kit, InVitrogen) and the nucleotide sequence was determined. A cDNA, showing high sequence homology with that encoding the human GM3 synthase from TPA-differentiated HL60 cells (3), has been successfully isolated and cloned from human placenta. The major difference between these two cDNAs is in the 5\u2019-end, according to the existence of different promoter regions, responsible for tissue-specific expression of the gene. Furthermore, the cDNA from the human placenta contains, upstream and in frame with the ATG indicated as translation initiation site for the GM3 synthase of HL60 cells, another ATG codon inserted in a sequence compatible with Kozak\u2019s rule, suggesting that the protein of the human placenta has an additional portion in NH2-terminus. The complete coding region of the human placenta cDNA is going to be cloned in an expression vector, under the control of the CMV promoter, in order to evaluate its activity. On the other hand, in vitro translation experiments are going to be carried out to define the first start codon. 1) Hakomori S.I. (2000): Glycoconj. J. 17, 627-647 2) Kolter T. et al. (2002): J.Biol.Chem. 277, 25859-25862 3) Ishii A. et al. (1998): J.B.C. 273, 31652-3165

IDENTIFICATION OF A NOVEL GM3 SYNTHASE mRNA ISOFORM FROM HUMAN PLACENTA AND FUNCTIONAL ANALYSIS OF THE 5\u2019-FLANKING REGION OF THE TRANSCRIPT

GM3 synthase is a key enzyme in the biosynthesis of gangliosides that, as mediators of cell-cell interactions and modulators of signaling transduction, play fundamental roles in many both physiological and pathological cellular processes (i.e., proliferation, differentiation, oncogenesis) (1,2). GM3 synthase results expressed in a tissue-specific manner, especially in brain, muscle, testis, and placenta, and only one transcript, of about 2.4 kb, has been detected in all the tissues. Up to date GM3 synthase cDNA has been isolated and characterized from both TPA-differentiated HL-60 cells and human fetal and adult brain (3,4) and five mRNA variants have been identified. They differ in the 5\u2019-untranslated region, but all of them predict a 41.7 kDa protein having substrate activity highly restricted to lactosylceramide. On the contrary, our study, directed to the isolation of the full length GM3 synthase cDNA from human placenta, led to the identification of an unusual GM3 synthase mRNA variant that strongly differs from all the other GM3 synthase transcripts until now identified. In fact, its 5\u2019-terminal region introduces a new translation initiation site located upstream and in-frame with that usually considered as translation initiation codon in the GM3 synthase gene. Studies of in vitro translation and in vivo expression indicate that the human placental cDNA leads to the synthesis of two polypeptides with apparent molecular mass, in SDS-PAGE, of 42.1 and 38.3 kDa, respectively. Moreover, the stable transfection of the human placental cDNA into mammalian MG1361 cells resulted in a threefold increase of GM3 synthase activity, associated to a broader substrate specificity, and, more importantly, in significant alterations of cell ganglioside profile. Therefore, our data provide the first demonstration of the existence of two GM3 synthase isoforms and indicate that, at least in the human placenta, they apparently derive from a single transcript. In addition, to elucidate the mechanism that regulates the tissue-specific expression of the human GM3 synthase gene we isolated the genomic DNA region (~3 kb) flanking the placental GM3 synthase transcript. Sequence analysis with the MatInspector 2.2 program revealed that this region lacks canonical TATA e CAAT boxes, but contains several putative transcription factor binding sites including AP1, GATA1, NFAT, MZF1, IK2; unfortunately no placenta-specific transcription factor is reported in none of the available transcription factor databases. Up to date, functional analysis of the isolated genomic region has been assessed in human placental choriocarcinoma JEG3 cells by transient transfection, but preliminary data seem to indicate that it doesn\u2019t promote transcription of the reporter SEAP gene. Future plans are to analyze the functional activity of DNA fragments containing varying lengths of the 5\u2019-flanking genomic sequence in both JEG3 cells and other suitable cell lines so to evaluate the eventual presence of positive and negative regulatory elements and factors in both the DNA sequence and the cell line. 1) Hakomori S.I. (2000) Glycoconj. J. 17, 143. 2) Hakomori S.I. (2000) Glycoconj. J. 17, 627. 3) Ishii A. et al. (1998) J. Biol. Chem. 273, 31652. 4) Kapitonov D. et al.(1999) Glycoconj J. 16, 337

GM3 IN THE REGULATION OF THE EXPRESSION AND ACTIVATION OF ErbB-2/EGFR HETERODIMERS

Gangliosides are a large and heterogeneous family of sialic acid containing glycosphingolipids, ubiquitous components of all eukaryotic cell membranes. They can partially segregate, together with cholesterol and specific signaling transduction proteins, such as receptor tyrosine-kinases, into unique more or less stable clusters or microdomains, indicated as \u201cglycolipid-enriched domains\u201d, \u201clipid rafts\u201d, \u201ccaveolae\u201d, contributing to the membrane structure, organization and function (1). Gangliosides are well-characterized modulators of receptor tyrosine-kinase (RTKs) phophorylation and dimerization (2). Our interest is particularly directed to study the relationship between gangliosides and two members of the tyrosine kinase ErbB receptor family: the epidermal growth factor receptor, EGFR or ErbB-1, and the structurally related protein ErbB-2. Unlike EGFR, ErbB-2 is a ligandless receptor: it can be activated by heterodimerization and cross-phosphorylation by other ligand-activated ErbB receptors (3,4). Our previous experimental evidence supports the functional relationship between ErbB-2 and gangliosides, in particular GM3 (5,6). In the present study, using the HC11 mouse mammary epithelial cell line, we investigated the ErbB-2 activation state and its tendency to form stable molecular complexes with EGFR and with ganglioside GM3, before and after EGF stimulation, by co-immunoprecipitation experiments with anti-ErbB-2 antibody and Western blot analyses. As HC11 cells express different ganglioside species, the exclusive association of GM3 with ErbB-2 and EGFR was ascertained by high performance-thin layer chromatography (HP-TLC) and TLC-immunostaining analyses of gangliosides extracted from the immunoprecipitates. Results display that in EGF-stimulated HC11 cells stable and tyrosine-phosphorylated ErbB2/EGFR dimers are formed and that GM3 is the unique ganglioside tightly associated to ErbB-2/EGFR dimers and to EGFR monomers, but not to ErbB2 monomers. In cells not stimulated with EGF a spontaneous but unproductive dimerization of ErbB2 and EGFR takes place and no ganglioside is found associated to the receptors. These observations indicate that the modulation of ErbB2 activation by GM3 may be mediated by EGFR, but that EGF stimulation is indispensable. After ganglioside depletion by [D]-PDMP, phosphorylated EGFR monomers are observed both before and after EGF stimulation, whereas ErbB-2 is present in the monomeric and unphosphorylated state even after EGF stimulation, suggesting that GM3 might have a bivalent key role in modulating the activation of ErbB-2 and EGFR. References 1. Fivaz, M., Abrami, L., Van Der Goot. F.G. Trends Cell Biol. 9(6), 212-213 (1999); 2. Bremer, E.G., Current topics in membranes 40, 387-411(1999); 3. Qian, X., LeVea, C.M., Freeman, J.K., Dougall, W.C., Greene, M.I., Proc. Natl. Acad. Sci. USA 91, 1500-1504 (1994); 4. Gulliford, T.J., Huang, G.C., Ouyang, X., Epstein, R.J., Oncogene 15, 2219-2223 (1997); 5. Sottocornola E., Berra B., and Colombo I., Biochim. Biophys. Acta-Molecolar and cell Biology of Lipids, 1635, 55-66 (2003); 6. Sottocornola E., Misasi R., Mattei V., Ciarlo L., Gradini R., Garofalo T., Berra B., Colombo I., and Sorice M., FEBS J. 273, 1821-1830 (2006)

Palladium-catalyzed domino carbopalladation/5-exo-allylic amination of \u3b1-amino allenamides: an efficient entry to enantiopure imidazolidinones

Allenamides of alpha-amino acids were converted into enantiopure 2-vinylimidazolidin-4-ones by a carbopalladation/exo-cyclization process. The products were obtained in 2.5:1-5.5:1 dr, with 94-99% ee. The palladium-catalyzed carbonylative cyclization of the same substrates afforded enone structures. Starting from properly substituted allenamides, an intramolecular carbopalladation followed by intramolecular amination gave rise to tricyclic fused-ring imidazolidinones