title: Direct inhibitory effects of a somatostatin analog, SMS 201-995, on AR4-2J cell proliferation via pertussis toxin-sensitive guanosine triphosphate-binding protein-independent mechanism.
abstract: Somatostatin has been demonstrated to negatively regulate pancreatic growth in vivo. In this study we used the AR4-2J rat pancreatic acinar tumor cell line to investigate the effect of a stable somatostatin analog, SMS 201-995 (SMS) on cell proliferation. SMS induced an antiproliferative effect on both serum or epidermal growth factor (EGF)-induced cell proliferation; exposure of the cells for 48 h to SMS caused a slight inhibition of serum-induced proliferation (maximal inhibition, 26%) and abolished the growth-promoting effect of EGF. Maximal effect was observed with 10 nM SMS, and half-maximal (IC50) effect with 0.06-0.1 nM SMS. Binding studies with an iodinated derivative of SMS, [125I-Tyr3]SMS, revealed the presence of a single class of high affinity binding sites on AR4-2J plasma membranes with an equilibrium dissociation constant of 0.2 +/- 0.03 nM and a binding site number of 1.1 +/- 0.07 pmol/mg protein. Addition of the nonhydrolyzable GTP analog, guanosine 5-[gamma-thio] triphosphate (GTP gamma S), increased the rate of dissociation of the specifically bound peptide in agreement with the coupling of somatostatin receptors with a GTP-binding regulatory protein. The good agreement between the IC50 for SMS inhibition of cell proliferation and the apparent Kd for binding indicates that the characterized binding sites are the somatostatin receptors that mediate the antiproliferative effect of SMS. When cells were grown in serum-free medium EGF stimulated AR4-2J cell proliferation with half-maximal (ED50) and maximal effects at 0.6 and 10 nM EGF, respectively. This stimulatory effect of EGF was mediated by specific receptors, since binding studies with [125I]EGF indicated that AR4-2J cells contained a single class of EGF receptors (13,000 sites/cell), with an affinity constant for [125I]EGF (Kd = 0.9 +/- 0.09 nM) close to the ED50 for EGF stimulation of cell growth. To examine if SMS-induced growth inhibition involved a cAMP-dependent mechanism we first studied the effect of SMS on cAMP production. SMS had no effect on basal cAMP, but completely inhibited VIP-stimulated cAMP production with an IC50 of 0.2 nM. Pertussis toxin, which is known to abolish the inhibitory effect of somatostatin on adenylate cyclase activity in AR4-2J cells, did not reverse the ability of SMS to inhibit cell proliferation as well as EGF-induced cell proliferation. These data indicate that the antiproliferative effect of SMS does not involve the GTP-binding protein-mediated negative coupling of somatostatin receptors to adenylate cyclase.(ABSTRACT TRUNCATED AT 400 WORDS)
authors.affiliation: INSERM U 151, CHU Rangueil, Toulouse, France.
authors.forename: N
authors.initials: N
authors.lastname: Viguerie
authors.forename: N
authors.initials: N
authors.lastname: Tahiri-Jouti
authors.forename: A M
authors.initials: AM
authors.lastname: Ayral
authors.forename: C
authors.initials: C
authors.lastname: Cambillau
authors.forename: J L
authors.initials: JL
authors.lastname: Scemama
authors.forename: M J
authors.initials: MJ
authors.lastname: Bastié
authors.forename: S
authors.initials: S
authors.lastname: Knuhtsen
authors.forename: J P
authors.initials: JP
authors.lastname: Estève
authors.forename: L
authors.initials: L
authors.lastname: Pradayrol
authors.forename: C
authors.initials: C
authors.lastname: Susini
mesh_terms: D037361:Adenylate Cyclase Toxin; D000818:Animals; D002455:Cell Division / Q000187:drug effects*; D002460:Cell Line; D000242:Cyclic AMP / Q000096:biosynthesis; D004261:DNA Replication / Q000187:drug effects; D004815:Epidermal Growth Factor / Q000494:pharmacology; D066246:ErbB Receptors / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000502:physiology*; D007700:Kinetics; D015282:Octreotide / Q000494:pharmacology*; D010190:Pancreatic Neoplasms; D037342:Pertussis Toxin; D017981:Receptors, Neurotransmitter / Q000378:metabolism; D017481:Receptors, Somatostatin; D013004:Somatostatin / Q000037:antagonists & inhibitors* / Q000378:metabolism / Q000494:pharmacology; D013936:Thymidine / Q000378:metabolism; D010566:Virulence Factors, Bordetella / Q000494:pharmacology*
chemical_list: D037361:Adenylate Cyclase Toxin; D017981:Receptors, Neurotransmitter; D017481:Receptors, Somatostatin; D010566:Virulence Factors, Bordetella; D013004:Somatostatin; D004815:Epidermal Growth Factor; D000242:Cyclic AMP; D037342:Pertussis Toxin; D066246:ErbB Receptors; D019204:GTP-Binding Proteins; D015282:Octreotide; D013936:Thymidine