title: Concentrative influx of functionally active cyclic ADP-ribose in dimethyl sulfoxide-differentiated HL-60 cells.
abstract: Native human HL-60 cells do not express CD38, a multifunctional ectoenzyme, which generates cyclic ADP-ribose (cADPR), a potent calcium mobilizer. However, when HL-60 cells are induced to differentiate to granulocytes by treatment with retinoic acid (RA), they express CD38 and accumulate cADPR. Both processes play a causal role in RA-induced differentiation. Other granulocyte differentiation-inducers, including dimethyl sulfoxide (Me(2)SO), fail to induce CD38 expression. We investigated whether treatment of HL-60 cells with Me(2)SO involves any changes in the cADPR/intracellular calcium ([Ca(2+)](i)) signaling system and, specifically, whether Me(2)SO affects those nucleoside transporters (NT) (both equilibrative (ENT) and concentrative (CNT)) that mediate influx of extracellular cADPR. Semiquantitative polymerase chain reaction analysis of transcripts, binding of [(3)H]nitrobenzylthioinosine (NBMPR) to intact cells, and influx experiments of extracellular cADPR (with selective inhibitors of NT as NBMPR or in specific conditions) were performed in native and Me(2)SO-differentiated HL-60 cells. The native cells showed uptake of cADPR across ENT2, whereas influx of cADPR into the Me(2)SO-differentiated cells occurred mostly by concentrative processes mediated by CNT3 and by an NBMPR-inhibitable concentrative NT designated cs-csg. Me(2)SO-differentiated, but not native HL-60 cells, accumulated cADPR and showed increased [Ca(2+)](i) levels when grown in a transwell co-culture setting over CD38-transfected 3T3 fibroblasts where nanomolar cADPR concentrations are present in the medium. NBMPR inhibited both responses of Me(2)SO-induced cells. Thus, concentrative influx of extracellular cADPR across CNT3 and cs-csg NT could substitute in the absence of CD38 in eliciting cADPR-dependent [Ca(2+)](i) increases in granulocyte-differentiated HL-60 cells, as well as in other CD38(-) cells.
authors.affiliation: Department of Experimental Medicine, University of Genova, Viale Benedetto XV/1, Genova, Italy.
authors.forename: Lucrezia
authors.initials: L
authors.lastname: Guida
authors.forename: Luisa
authors.initials: L
authors.lastname: Franco
authors.forename: Santina
authors.initials: S
authors.lastname: Bruzzone
authors.forename: Laura
authors.initials: L
authors.lastname: Sturla
authors.forename: Elena
authors.initials: E
authors.lastname: Zocchi
authors.forename: Giovanna
authors.initials: G
authors.lastname: Basile
authors.forename: Cesare
authors.initials: C
authors.lastname: Usai
authors.forename: Antonio
authors.initials: A
authors.lastname: De Flora
mesh_terms: D036541:ADP-ribosyl Cyclase / Q000096:biosynthesis; D051997:ADP-ribosyl Cyclase 1; D000818:Animals; D015703:Antigens, CD / Q000096:biosynthesis; D001692:Biological Transport; D019556:COS Cells; D002118:Calcium / Q000737:chemistry; D002454:Cell Differentiation; D018920:Coculture Techniques; D036563:Cyclic ADP-Ribose / Q000737:chemistry*; D018076:DNA, Complementary / Q000378:metabolism; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D018922:HL-60 Cells; D006801:Humans; D007700:Kinetics; D008562:Membrane Glycoproteins; D051379:Mice; D041681:NIH 3T3 Cells; D011485:Protein Binding; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D013997:Time Factors; D014162:Transfection; D014212:Tretinoin / Q000378:metabolism
chemical_list: D015703:Antigens, CD; D018076:DNA, Complementary; D008562:Membrane Glycoproteins; D012333:RNA, Messenger; D036563:Cyclic ADP-Ribose; D014212:Tretinoin; D036541:ADP-ribosyl Cyclase; C498154:CD38 protein, human; C498155:Cd38 protein, mouse; D051997:ADP-ribosyl Cyclase 1; D002118:Calcium; D004121:Dimethyl Sulfoxide