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• W2017235467 abstract "Activation of the somatostatin receptor sst2 inhibits cell proliferation by a mechanism involving the stimulation of the protein-tyrosine phosphatase SHP-1. The cell cycle regulatory events leading to sst2-mediated growth arrest are not known. Here, we report that treatment of Chinese hamster ovary cells expressing sst2 with the somatostatin analogue, RC-160, led to G1cell cycle arrest and inhibition of insulin-induced S-phase entry through induction of the cyclin-dependent kinase inhibitor p27 Kip1. Consequently, a decrease of p27 Kip1-cdk2 association, an inhibition of insulin-induced cyclin E-cdk2 kinase activity, and an accumulation of hypophosphorylated retinoblastoma gene product (Rb) were observed. However, RC-160 had no effect on the p21 Waf1/Cip1. When sst2 was coexpressed with a catalytically inactive mutant SHP-1 in Chinese hamster ovary cells, mutant SHP-1 induced entry into cell cycle and down-regulation of p27 Kip1and prevented modulation by insulin and RC-160 of p27 Kip1expression, p27 Kip1-cdk2 association, cyclin E-cdk2 kinase activity, and the phosphorylation state of Rb. In mouse pancreatic acini, RC-160 reverted down-regulation of p27 Kip1 induced by a mitogen, and this effect did not occur in acini from viable motheaten (me v /me v) mice expressing a mutant SHP-1 with markedly deficient enzymes. These findings provide the first evidence that sst2 induces cell cycle arrest through the up-regulation of p27 Kip1 and demonstrate that SHP-1 is required for maintaining high inhibitory levels of p27 Kip1and is a critical target of the insulin, and somatostatin signaling cascade, leading to the modulation of p27 Kip1. Activation of the somatostatin receptor sst2 inhibits cell proliferation by a mechanism involving the stimulation of the protein-tyrosine phosphatase SHP-1. The cell cycle regulatory events leading to sst2-mediated growth arrest are not known. Here, we report that treatment of Chinese hamster ovary cells expressing sst2 with the somatostatin analogue, RC-160, led to G1cell cycle arrest and inhibition of insulin-induced S-phase entry through induction of the cyclin-dependent kinase inhibitor p27 Kip1. Consequently, a decrease of p27 Kip1-cdk2 association, an inhibition of insulin-induced cyclin E-cdk2 kinase activity, and an accumulation of hypophosphorylated retinoblastoma gene product (Rb) were observed. However, RC-160 had no effect on the p21 Waf1/Cip1. When sst2 was coexpressed with a catalytically inactive mutant SHP-1 in Chinese hamster ovary cells, mutant SHP-1 induced entry into cell cycle and down-regulation of p27 Kip1and prevented modulation by insulin and RC-160 of p27 Kip1expression, p27 Kip1-cdk2 association, cyclin E-cdk2 kinase activity, and the phosphorylation state of Rb. In mouse pancreatic acini, RC-160 reverted down-regulation of p27 Kip1 induced by a mitogen, and this effect did not occur in acini from viable motheaten (me v /me v) mice expressing a mutant SHP-1 with markedly deficient enzymes. These findings provide the first evidence that sst2 induces cell cycle arrest through the up-regulation of p27 Kip1 and demonstrate that SHP-1 is required for maintaining high inhibitory levels of p27 Kip1and is a critical target of the insulin, and somatostatin signaling cascade, leading to the modulation of p27 Kip1. Somatostatin is a widely distributed inhibitory hormone that plays an important role in several biological processes including neurotransmission, inhibition of exocrine and endocrine secretions, and cell proliferation. The diverse biological effects of somatostatin are mediated through a family of five somatostatin receptors (sst1-sst5) that belong to the family of G-protein-coupled receptors and that regulate diverse signal transduction pathways including adenylate cyclase, phospholipase C-β, phospholipase A2, guanylate cyclase, ionic conductance channels, and tyrosine phosphatase (1Lewin M.J. Annu. Rev. Physiol. 1992; 54: 455-468Crossref PubMed Google Scholar,2Patel Y.C. J. Endocrinol. Invest. 1997; 20: 348-367Crossref PubMed Scopus (271) Google Scholar). The ability of somatostatin and its stable analogues to promote inhibition of normal and tumor cell growth has been demonstrated in various cell types including mammary, prostatic, gastric, pancreatic, colorectal, and small cell lung cancer cells (3Liebow C. Reilly C. Serrano M. Schally A.V. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 2003-2007Crossref PubMed Scopus (308) Google Scholar, 4Lamberts S.W. Krenning E.P. Reubi J.C. Endocr. Rev. 1991; 12: 450-482Crossref PubMed Scopus (848) Google Scholar). However, the mechanisms of cell growth arrest by somatostatin are still poorly understood. Somatostatin analogues induce a G0/G1 cell cycle arrest and thus prevent DNA synthesis in GH3 rat pituitary tumor cells, whereas they induce a transient G2/M blockade as well as apoptosis in MCF7 human mammary tumor cells (5Cheung N.W. Boyages S.C. Endocrinology. 1995; 136: 4174-4181Crossref PubMed Scopus (0) Google Scholar, 6Pagliacci M.C. Tognellini R. Grignani F. Nicoletti I. Endocrinology. 1991; 129: 2555-2562Crossref PubMed Scopus (86) Google Scholar). These tumor cells express multiple somatostatin receptors and the question of whether different somatostatin receptor(s) may be involved in eliciting these effects still remains to be clarified. A specific role for sst3 in transducing apoptosis through an induction of p53 and Bax has been reported (7Sharma K. Patel Y.C. Srikant C.B. Mol. Endocrinol. 1996; 10: 1688-1696Crossref PubMed Scopus (255) Google Scholar). Control of the cell cycle machinery by other receptors is an important problem that remains to be addressed. Our studies on the expression of somatostatin receptor subtypes in heterologous systems led us to demonstrate that sst2 selectively mediates the antiproliferative effect of somatostatin analogues on serum- or insulin-induced cell growth through the stimulation of a protein-tyrosine phosphatase (8Buscail L. Estève J.-P. Saint-Laurent N. Bertrand V. Reisine T.A.M.O.C. Bell G.I. Schally A.V. Vaysse N. Susini C. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1580-1584Crossref PubMed Scopus (298) Google Scholar), which was recently identified as SHP-1 (9Lopez F. Estève J.-P. Buscail L. Delesque N. Saint-Laurent N. Theveniau M. Nahmias C. Vaysse N. Susini C. J. Biol. Chem. 1997; 272: 24448-24454Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 10Bousquet C. Delesque N. Lopez F. Saint-Laurent N. Estève J.-P. Bedecs K. Buscail L. Vaysse N. Susini C. J. Biol. Chem. 1998; 273: 7099-7106Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). SHP-1, a protein-tyrosine phosphatase with two SH2 domains, plays a role in terminating growth factor and cytokine signals by dephosphorylating critical molecules (reviewed in Ref. 11Feng G.S. Pawson T. Trends Genet. 1994; 10: 54-58Abstract Full Text PDF PubMed Scopus (169) Google Scholar). We reported that SHP-1 is activated by somatostatin and participates in the negative regulation of mitogenic insulin signaling as a result of its association with and dephosphorylation of insulin receptor as well as associated molecules (9Lopez F. Estève J.-P. Buscail L. Delesque N. Saint-Laurent N. Theveniau M. Nahmias C. Vaysse N. Susini C. J. Biol. Chem. 1997; 272: 24448-24454Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 10Bousquet C. Delesque N. Lopez F. Saint-Laurent N. Estève J.-P. Bedecs K. Buscail L. Vaysse N. Susini C. J. Biol. Chem. 1998; 273: 7099-7106Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). However, the effect of sst2 as well as the role of SHP-1 on cell cycle parameters remain unknown. Cell cycle progression is dependent on the coordinated interaction, posttranslational modification, and degradation of cyclins and their catalytic partners, cyclin-dependent kinases (cdks). 1The abbreviations used are: cdk, cyclin-dependent kinase; Rb, retinoblastoma gene product; pRb, phosphorylated Rb; CHO, chinese hamster ovary; αMEM, minimal essential medium; PAGE, polyacrylamide gel electrophoresis; EGF, epidermal growth factor. Cyclins are expressed at particular stages of the cell cycle and associate with specific cdks to form active complexes that phosphorylate multiple proteins and promote cell cycle progression. In mammalian cells, progression through early to middle G1 phase of the cell cycle is dependent on cdk4/and/or cdk6, which are activated by D-type cyclins. Transition through middle G1 to S phase is regulated by activation of cdk2 by cyclin E, cdk2, and cyclin A is required for late G1 to S-phase progression and throughout S phase. One of the critical targets of cyclin-cdk complexes is the retinoblastoma gene product (Rb). Rb acts as a transcriptional repressor. In its hypophosphorylated form, it binds to the E2F family of cell cycle transcription factors during G1 phase and inhibits E2F activity. Rb is inactivated by cdk phosphorylation in mid to late G1 phase of the cell cycle and dissociates from E2F, leading to activation of genes containing E2F sites and a progression from G1 to S phase (reviewed in Ref. 12Sherr C.J. Roberts J.M. Genes Dev. 1995; 9: 1149-1163Crossref PubMed Scopus (3221) Google Scholar). Another level of regulation of cdk activity results from the action of cdk inhibitors that bind cyclin-cdk complexes and either inhibit their kinase activities or prevent their activation by cdk-activating kinase (reviewed in Refs. 13Sherr C.J. Trends Biochem. Sci. 1995; 20: 187-190Abstract Full Text PDF PubMed Scopus (890) Google Scholar and 14Morgan D.O. Nature. 1995; 374: 131-134Crossref PubMed Scopus (2938) Google Scholar). In mammalian cells, cdk inhibitors comprise two classes of proteins, the Ink4 family including p16 Ink4a, p15 Ink4b, p18 Ink4c, and p19 Ink4d, which specifically inhibit cyclin D-dependent kinases, cdk4 and cdk6 (12Sherr C.J. Roberts J.M. Genes Dev. 1995; 9: 1149-1163Crossref PubMed Scopus (3221) Google Scholar), and the p21 family including p21 Cip1/Waf1, p27 Kip1, and p57 Kip2(15Harper J.W. Adami G.R. Wei N. Keyomarsi K. Elledge S.J. Cell. 1993; 75: 805-816Abstract Full Text PDF PubMed Scopus (5250) Google Scholar, 16Xiong Y. Hannon G.J. Zhang H. Casso D. Kobayashi R. Beach D. Nature. 1993; 366: 701-704Crossref PubMed Scopus (3179) Google Scholar, 17Polyak K. Lee M.H. Erdjument-Bromage H. Koff A. Roberts J.M. Tempst P. Massague J. Cell. 1994; 78: 59-66Abstract Full Text PDF PubMed Scopus (2057) Google Scholar, 18Toyoshima H. Hunter T. Cell. 1994; 78: 67-74Abstract Full Text PDF PubMed Scopus (1938) Google Scholar, 19Lee M.H. Reynisdottir I. Massague J. Genes Dev. 1995; 9: 639-649Crossref PubMed Scopus (856) Google Scholar), which can interact with many different cyclin-cdk complexes. Among them, p27 Kip1 is a widely distributed cdk inhibitor that has an important role regulating entry into and exit from the cell cycle. p27 Kip1 is abundantly expressed in normal quiescent cells and is down-regulated by mitogens. The decrease in p27 Kip1 expression occurs through protein degradation via the ubiquitin-proteasome pathway after p27 Kip1 phosphorylation by cyclin E-cdk2 complexes (20Pagano M. Tam S.W. Theodoras A.M. Beer-Romero P. Del Sal G. Chau V. Yew P.R. Draetta G.F. Rolfe M. Science. 1995; 269: 682-685Crossref PubMed Scopus (1736) Google Scholar, 21Sheaff R.J. Groudine M. Gordon M. Roberts J.M. Clurman B.E. Genes Dev. 1997; 11: 1464-1478Crossref PubMed Scopus (797) Google Scholar, 22Vlach J. Hennecke S. Amati B. EMBO J. 1997; 16: 5334-5344Crossref PubMed Scopus (609) Google Scholar). Increased levels of p27 Kip1induced by transforming growth factor β, contact inhibition, serum deprivation, rapamycin, or staurosporine have been associated with a G1 arrest (18Toyoshima H. Hunter T. Cell. 1994; 78: 67-74Abstract Full Text PDF PubMed Scopus (1938) Google Scholar, 23Polyak K. Kato J.Y. Solomon M.J. Sherr C.J. Massague J. Roberts J.M. Koff A. Genes Dev. 1994; 8: 9-22Crossref PubMed Scopus (1837) Google Scholar, 24Kwon T.K. Buchholz M.A. Chrest F.J. Nordin A.A. Cell Growth Differ. 1996; 7: 1305-1313PubMed Google Scholar). In contrast, an overexpression of p27 Kip1 antisense cDNA results in mitogen-independent G1 progression, demonstrating the importance of p27 Kip1 in controlling cell cycle exit (25Rivard N. L'Allemain G. Bartek J. Pouyssegur J. J. Biol. Chem. 1996; 271: 18337-18341Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar, 26Coats S. Flanagan W.M. Nourse J. Roberts J.M. Science. 1996; 272: 877-880Crossref PubMed Scopus (652) Google Scholar). The involvement of p27 Kip1 in the negative regulation of cell proliferation is related to its binding and subsequent inhibition of the kinase activity of cdk2-cyclin complexes (17Polyak K. Lee M.H. Erdjument-Bromage H. Koff A. Roberts J.M. Tempst P. Massague J. Cell. 1994; 78: 59-66Abstract Full Text PDF PubMed Scopus (2057) Google Scholar, 18Toyoshima H. Hunter T. Cell. 1994; 78: 67-74Abstract Full Text PDF PubMed Scopus (1938) Google Scholar, 23Polyak K. Kato J.Y. Solomon M.J. Sherr C.J. Massague J. Roberts J.M. Koff A. Genes Dev. 1994; 8: 9-22Crossref PubMed Scopus (1837) Google Scholar, 27Kwon T.K. Buchholz M.A. Nordin A.A. Biochem. Biophys. Res. Commun. 1996; 220: 703-709Crossref PubMed Scopus (9) Google Scholar). In this study, we investigated the potential effects of sst2 on cell cycle progression and expression of cell cycle regulatory proteins in CHO cells expressing sst2 (CHO/sst2). Activation of sst2 caused a G1 cell cycle arrest in-phase accompanied by an increased expression of cdk inhibitor p27 Kip1, which resulted in an increase of its association with cdk2 and a decrease in cdk2 activity and led to dephosphorylation of protein Rb. The role of SHP-1 in sst2-mediated regulatory mechanisms was investigated in CHO cells coexpressing sst2 and a negative SHP-1 (C453S) mutant as well as in acini isolated from viable motheaten mice expressing a mutant SHP-1 with markedly deficient enzyme activity. Our results provide evidence that SHP-1 is a critical regulator of p27 Kip1. This enzyme is required for the maintenance of cell quiescence and is involved in the sst2-mediated up-regulation of p27 Kip1 leading to cell cycle arrest. Monoclonal anti-p27 Kip1 and anti-Rb antibodies, known to react with murine and human proteins, were purchased from Transduction Laboratories and Pharmingen, respectively. Monoclonal anti-human p21 Waf1/Cip1 antibodies were from Transduction Laboratories. Polyclonal anti-mouse p21 Waf1/Cip1and anti-cdk2 antibodies that react with human and murine proteins were from Santa Cruz Biotechnology. Monoclonal anticyclin E antibodies react with human and murine proteins and were from Calbiochem. RC-160 was synthesized as described previously (28Cai R.Z. Szoke B. Lu R. Fu D. Redding T.W. Schally A.V. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 1896-1900Crossref PubMed Scopus (182) Google Scholar). [γ33P]ATP (3,000 Ci/mmol) was purchased from Isotopchim (France), histone H1 was from Sigma, and the enhanced chemiluminescence (ECL) immunodetection system was from Amersham Pharmacia Biotech. The 1.2-kilobase XbaI fragment of mouse sst2A cDNA subcloned into pCMV6c vector was stably co-transfected in CHO (DG44 variant) cells using Lipofectin reagent with pSV2neo as described (kindly donated by Dr. G. I. Bell, Howard Hughes Medical Institute, University of Chicago and Dr. T. Reisine, University of Pennsylvania, School of Medicine, Philadelphia) (8Buscail L. Estève J.-P. Saint-Laurent N. Bertrand V. Reisine T.A.M.O.C. Bell G.I. Schally A.V. Vaysse N. Susini C. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1580-1584Crossref PubMed Scopus (298) Google Scholar). Stable transfectants were selected in αMEM (minimal essential medium) containing geneticin at 600 μg/ml. Geneticin-resistant clones expressing sst2 (CHO/sst2) were screened for somatostatin binding using [125I-Tyr11] somatostatin as tracer as described (8Buscail L. Estève J.-P. Saint-Laurent N. Bertrand V. Reisine T.A.M.O.C. Bell G.I. Schally A.V. Vaysse N. Susini C. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1580-1584Crossref PubMed Scopus (298) Google Scholar). The 2.1-kilobase HindIII/NotI fragment of human SHP-1 cDNA (a gift of Dr. M. L. Thomas, Howard Hughes Medical Institute, Washington University, St. Louis, MO) was subcloned into the expression vector pcDNA I neo vector (Invitrogen). The SHP-1 (C453S) mutant (a gift of Dr. C. Nahmias, ICGM, Paris) was constructed as described (10Bousquet C. Delesque N. Lopez F. Saint-Laurent N. Estève J.-P. Bedecs K. Buscail L. Vaysse N. Susini C. J. Biol. Chem. 1998; 273: 7099-7106Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). The mouse sst2 gene in the pCMV6c vector was stably co-transfected in CHO cells using Lipofectin reagent with the SHP-1 (C453S) mutant in pcDNA I neo. Stable colonies obtained by selection with G418 (600 μg/ml) were screened for somatostatin binding and the presence of SHP-1 as described (9Lopez F. Estève J.-P. Buscail L. Delesque N. Saint-Laurent N. Theveniau M. Nahmias C. Vaysse N. Susini C. J. Biol. Chem. 1997; 272: 24448-24454Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). CHO-DG44 stably expressing sst2 (CHO/sst2) or sst2 and SHP-1 (C453S) (CHO/sst2-SHP-1(C453S)) were cultured in αMEM containing 10% fetal calf serum and G418 (200 μg/ml) as described previously (8Buscail L. Estève J.-P. Saint-Laurent N. Bertrand V. Reisine T.A.M.O.C. Bell G.I. Schally A.V. Vaysse N. Susini C. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1580-1584Crossref PubMed Scopus (298) Google Scholar). After an overnight attachment phase, cells were serum-starved in αMEM for 18 h without geneticin before peptide addition. C57BL6-me v /me vmice were obtained by mating heterozygous C57BL6-me v/+ mice (Jackson Laboratories, Bar Harbor, ME) breeding pairs. Homozygousme v /me v were screened by reverse transcription-polymerase chain reaction as described (29Shultz L.D. Schweitzer P.A. Rajan T.V. Yi T. Ihle J.N. Matthews R.J. Thomas M.L. Beier D.R. Cell. 1993; 73: 1445-1454Abstract Full Text PDF PubMed Scopus (690) Google Scholar).Me v /me v were identifiable by 10–15 days of age, because of the motheaten appearance of the skin.Me v /me v or their unaffected littermates were sacrificed at 3 weeks of age, and pancreases were removed. Pancreatic acini were prepared using enzymatic digestion of the pancreas with 1.5 mg/ml collagenase in an oxygenated Krebs-Ringer buffer containing 0.2% bovine serum albumin and 0.01% soybean trypsin inhibitor as described previously (buffer A) (30Knuhtsen S. Estève J.-P. Cambillau C. Colas B. Susini C. Vaysse N. J. Biol. Chem. 1990; 265: 1129-1133Abstract Full Text PDF PubMed Google Scholar). After washing with buffer A, acinar cells were incubated in the same buffer in the presence or absence of peptides at 25 °C for indicated times. Acini were then transferred to 0.3 m sucrose, sedimented at 800 × g for 5 min at 4 °C, and solubilized in lysis buffer (50 mm HEPES, 150 mm NaCl, 10 mm EDTA, 10 mmNa4P2O7, 100 mm NaF, 2 mm sodium orthovanadate (pH 7.4)) (buffer B) containing 0.1% Triton X-100, 1 mm benzamidine, and 0.01% soybean trypsin inhibitor at 4 °C for 30 min. Lysates were collected and centrifuged at 13,000 × g for 10 min at 4 °C and used for immunoprecipitation or immunoblotting. Cells were harvested by trypsin (0.5 mg/ml) and EDTA (0.02 mg/ml), washed twice with phosphate-buffered saline (pH 7.4), and centrifuged at 800 × g for 5 min at 4 °C. Cells were then incubated in the presence of 200 μl of trypsin (30 mg/ml) for 10 min. 200 μl of RNase (0.1 mg/ml) and trypsin inhibitors (0.5 mg/ml) were added for 10 min, and cells were stained with 250 μl of propidium iodide solution (125 μg/ml) for at least 15 min at 4 °C. Fluorescence of labeled cell nuclei was measured by flow cytometry using a FACScan (Beckton Dickinson) with a minimum of 10,000 events performed for each sample. Data were analyzed using LYSIS II software. Cells were washed with phosphate-buffered saline and then with buffer B. Cells were lysed in 500 μl of buffer B containing 1% Triton X-100, 1 mmphenylmethylsulfonylfluoride, 20 μg/ml aprotinin, 20 μmleupeptin. After a 15-min incubation at 4 °C, the lysate was collected and centrifuged at 13,000 × g for 10 min at 4 °C. Soluble proteins (300–500 μg) were incubated for 3 h at 4 °C with specific antibodies or preimmune serum prebound to Sepharose-protein A beads prewashed in buffer B. The beads were then washed twice with buffer B and resuspended in sample buffer for immunoblotting. For immunoblotting, solubilized proteins or immunoprecipitated proteins (see above) were resolved on 7.5% or 12% SDS-polyacrylamide gels, transferred to a nitrocellulose membrane, and immunoblotted with specific antibodies as described previously (9Lopez F. Estève J.-P. Buscail L. Delesque N. Saint-Laurent N. Theveniau M. Nahmias C. Vaysse N. Susini C. J. Biol. Chem. 1997; 272: 24448-24454Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). Immunoreactive proteins were visualized by the ECL immunodetection system and quantified by image analysis using a Biocom apparatus (Biocom, Paris, France). Immunoprecipitated proteins with anti-cdk2 or anticyclin E antibodies were collected by centrifugation and washed four times with buffer B and twice with 50 mm HEPES buffer containing 1 mm dithiotreitol (pH 7.4). The beads were suspended in 40 μl of kinase buffer containing 50 mmHEPES (pH 7.5), 10 mm MgCl2, 1 mmdithiothreitol, 4 μg of histone H1, 2.5 mm EGTA, 0.1 mm sodium orthovanadate, 1 mm NaF, 50 μm ATP, and 10 μCi of [γ-33P]ATP (1000–3000 Ci/mmol) and incubated for 30 min at 25 °C. Each sample was mixed with 20 μl of 2× SDS sample buffer, heated for 5 min at 100 °C, and subjected to SDS-PAGE. The gel was fixed in 40% methanol, 10% acetic acid and exposed to hyperfilm ECL. We previously reported that in CHO cells expressing sst2, the addition of the somatostatin analogue, RC-160, for 24 h to the culture medium led to inhibition of the mitogenic effect of insulin (10Bousquet C. Delesque N. Lopez F. Saint-Laurent N. Estève J.-P. Bedecs K. Buscail L. Vaysse N. Susini C. J. Biol. Chem. 1998; 273: 7099-7106Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). To analyze whether RC-160-mediated inhibition of cell proliferation reflects a stage-specific arrest of the cell cycle, cells were rendered quiescent by serum deprivation and incubated with 100 nm insulin in the presence or absence of 1 nm RC-160 and then analyzed by flow cytometry. Cells grown in the absence of fetal calf serum were taken as control values. The treatment of cells with insulin increased the percentage of cells in the S phase, which reached 27% at 6 h and increased up to 50% at 24 h of treatment (data not shown). The simultaneous treatment of cells with insulin and RC-160 for 6 h prevented cells from entering into the S phase, RC-160 causing a decrease in the percentage of cells in the S phase (−43%) and an accumulation of cells in the G1 phase, which increased from 57% in the absence of RC-160 to 72% (Fig. 1). For longer treatment, RC-160 had no significant effect on the G1/S transition (data not shown). We concluded that activation of sst2 by ligand induces a G1 cell cycle arrest in CHO/sst2 cells. Furthermore, treatment of cells with 1 μm orthovanadate suppressed the RC 160-induced decrease of number of cells in the S phase as well as the increase of cells in the G1 phase, indicating that a tyrosine phosphatase was required in the RC-160 effects (data not shown). G1 progression depends on an orderly and coordinated expression of cyclins that bind to and activate cdks, the activity of which is negatively regulated by their association with a family of cdk inhibitory proteins. Therefore we investigated whether sst2-mediated cell cycle arrest is associated with a change in the expression of the cdk inhibitors. We first examined the expression of the Kip/Cip family cdk inhibitor p27 Kip1, which has been demonstrated to be involved in the regulation of cell cycle progression induced by various antiproliferative stimuli that cause G1-phase arrest (23Polyak K. Kato J.Y. Solomon M.J. Sherr C.J. Massague J. Roberts J.M. Koff A. Genes Dev. 1994; 8: 9-22Crossref PubMed Scopus (1837) Google Scholar, 24Kwon T.K. Buchholz M.A. Chrest F.J. Nordin A.A. Cell Growth Differ. 1996; 7: 1305-1313PubMed Google Scholar, 25Rivard N. L'Allemain G. Bartek J. Pouyssegur J. J. Biol. Chem. 1996; 271: 18337-18341Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar, 26Coats S. Flanagan W.M. Nourse J. Roberts J.M. Science. 1996; 272: 877-880Crossref PubMed Scopus (652) Google Scholar). CHO/sst2 cells were treated for various times with insulin in the presence or not of RC-160, and the level of p27 Kip1 was investigated by Western blot analysis. As observed in Fig. 2, p27 Kip1 was expressed at high level in growth-arrested control cells, and after 3 h of insulin treatment, its expression decreased by 45% (p < 0.05), consistent with previous results reported for mitogenic signals (26Coats S. Flanagan W.M. Nourse J. Roberts J.M. Science. 1996; 272: 877-880Crossref PubMed Scopus (652) Google Scholar, 31Mann D.J. Higgins T. Jones N.C. Rozengurt E. Oncogene. 1997; 14: 1759-1766Crossref PubMed Scopus (51) Google Scholar, 32Aktas H. Cai H. Cooper G.M. Mol. Cell. Biol. 1997; 17: 3850-3857Crossref PubMed Scopus (372) Google Scholar). The decrease of p27 Kip1 level was transient, the level of this cdk inhibitor being not significantly different from that in control cells by 24 h. The addition of RC-160 resulted in a 4-fold increase (p < 0.02) in the level of p27 Kip1 during the first 3 h. Elevated levels of p27 Kip1 were found to return to control levels at 24 h of treatment with RC-160. Treatment of cells with 1 μmorthovanadate for 3 h suppressed the RC 160-induced increase of p27 Kip1, indicating that this effect is dependent on a tyrosine phosphatase (Fig. 2). The expression of the other member of the Kip/Cip family cdk inhibitors, p21 Waf1/Cip1, was also examined in CHO/sst2 cells. In contrast to p27 Kip1, p21 Waf1/Cip1 was found to be barely detectable in control cells (Fig. 3), as observed by others in quiescent cells (33Akiyama T. Yoshida T. Tsujita T. Shimizu M. Mizukami T. Okabe M. Akinaga S. Cancer Res. 1997; 57: 1495-1501PubMed Google Scholar). As reported for mitogens in other cell systems (32Aktas H. Cai H. Cooper G.M. Mol. Cell. Biol. 1997; 17: 3850-3857Crossref PubMed Scopus (372) Google Scholar, 34Reynisdottir I. Polyak K. Iavarone A. Massague J. Genes Dev. 1995; 9: 1831-1845Crossref PubMed Scopus (893) Google Scholar), insulin induced an increase of its expression up to 24 h, suggesting that elevated p21 Waf1/Cip1 is not related to insulin-mediated G1/S transition. However, the addition of RC-160 did not significantly modify the insulin-induced expression of p21 Waf1/Cip1 irrespective of the time of treatment, suggesting that this inhibitor is not involved in the somatostatin-mediated growth arrest. It has been shown in many cell types that among the G1 cyclin-cdk complexes negatively regulated by p27 Kip1, the up-regulation of p27 Kip1 in response to growth inhibitory factors favors its association with cyclin E-cdk2, resulting in kinase inhibition and contributing to cell growth arrest (34Reynisdottir I. Polyak K. Iavarone A. Massague J. Genes Dev. 1995; 9: 1831-1845Crossref PubMed Scopus (893) Google Scholar). Therefore, we first tested whether somatostatin analogue-mediated increases in the level of p27 Kip1 should be reflected in a change of the kinase activity of cdk2-associated complexes, as measured by an in vitro assay on cdk2 immunoprecipitates using histone H1 protein as a substrate. In comparison with the control activity detected in resting CHO/sst2 cells, cdk2 kinase activity was increased by about 2-fold after 3 h of treatment with insulin, as revealed by the heavily phosphorylated histone H1 level. When RC-160 was added to the culture, the cdk2-dependent kinase activity was inhibited by 80% during the first 3 h of culture (Fig. 4A). Similarly, RC-160 induced a decrease of about 80% insulin-induced increase of cyclin E-cdk2 associated kinase activity (Fig. 4B). In addition, the amount of p27 Kip1 associated with cdk2 was decreased by 70% after treatment of cells for 3 h with insulin, whereas the addition of RC-160 increased the level of the complexes by 87% (Fig. 4C). These results indicate that insulin and RC-160 could contrarily modulate the level of cdk2-cyclin E complexes, free from the constraining influence of p27 Kip1 inhibitor and the resulting kinase activity of the complexes. We then analyzed the steady-state level of expression of cyclin E and cdk proteins by immunoblotting. As shown in Fig. 4D, the amount of cdk2 detected in control cells and in insulin-treated cells was not significantly modified by RC-160 treatment. Anticyclin E antibodies revealed that cyclin E was barely detectable in control cells, and treatment of cells for 3 h with insulin induced an increase of the level of cyclin E as observed for agents that promote DNA synthesis (31Mann D.J. Higgins T. Jones N.C. Rozengurt E. Oncogene. 1997; 14: 1759-1766Crossref PubMed Scopus (51) Google Scholar). The addition of RC-160 resulted in a 50% inhibition of cyclin E expression after 3 h of treatment. One of the targets of cdk includes pRb protein, as its hyperphosphorylated form was proven to be a critical check point involved in regulating progression through late G1 and into S phase. The extent of pRb phosphorylation was first analyzed in soluble extracts of CHO/sst2 cells incubated in the presence of 100 nm insulin with or without 1 nm RC-160 for various times. A specific antibody able to detect the active hypophosphorylated form of pRb (Fig. 5, lower band) as well as the inactive slower migrating hyperphosphorylated form o" @default.
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• W2017235467 title "sst2 Somatostatin Receptor Mediates Cell Cycle Arrest and Induction of p27" @default.
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• W2017235467 doi "https://doi.org/10.1074/jbc.274.21.15186" @default.
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