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Activation of Human Somatostatin Receptor Type 2 Causes Inhibition of Cell Growth in Transfected HEK293 but Not in Transfected CHO Cells
JOURNAL OF SURGICAL RESEARCH ARTICLE NO.
71, 13–18 (1997)
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Activation of Human Somatostatin Receptor Type 2 Causes Inhibition of Cell Growth in Transfected HEK293 but Not in Transfected CHO Cells J. Ren,* G. Bell,† D. H. Coy,‡ and F. C. Brunicardi*,1 *Department of Surgery, Baylor College of Medicine, Houston, Texas 77030; †Howard Hughes Medical Institute, University of Chicago, Chicago, Illinois 60637; and ‡Tulane University, New Orleans, Louisiana 70118 Presented at the Annual Meeting of the Association for Academic Surgery, Chicago, Illinois, November 13–16, 1996
acts via activation of somatostatin receptors [4–8]. There are two naturally occurring forms of bioactive somatostatin, the 14-amino-acid-containing somatostatin (SS-14) and its N-terminally extended precursor somatostatin-28 (SS-28) [8, 9]. The antiproliferative effect of somatostatin has been studied in vitro and in vivo, and this peptide may participate in the regulation of cell growth both in normal and tumor cells [10]. Recently, a family of somatostatin receptors (SSTR) has been cloned and referred to as SSTR1-5 [2, 3]. SSTRs are seven transmembrane domain guanine nucleotide binding protein (G-protein)-coupled receptors and are coupled to multiple second messenger systems. Mouse, human and rat SSTR 2 generate two separate isoforms, a long (SSTR2A) and a short (SSTR2B) variant through alternate mRNA splicing at the C-terminal portion [11–13]. mRNAs for the five receptor subtypes are expressed in a tissue- and species-specific manner. Human SSTR1-5 mRNA is expressed in the brain, jejunum, stomach, pancreatic islets, and lung [14–18]. mRNA expression of both rat SSTR1-5 and mouse SSTR1-3 are found in the brain [14, 19–23]. The rat stomach expresses the four subtypes except SSTR5, while the liver and pancreas express only SSTR3 and SSTR2, respectively [20]. The distribution of somatostatin receptor type 2B is detected in brain and pituitary of rat and mouse, human pituitary tumors, and a human small cell lung cancer cell line [11–13]. The abundance of SSTR2B is higher than that of SSTR2A in mouse brain [11]. SSTR1-4 show similar high affinity with somatostatin-14 and somatostatin-28 [14–17]. On the other hand, rSSTR5 and hSSTR5 exhibit the higher affinity for somatostatin-28 than somatostatin-14 [16, 18, 24, 25]. We previously demonstrated that insulin release from human islets was inhibited by SSTR2 agonist and that the human pancreatic b cell was primarily occupied by SSTR2 [26]. Since mechanism of human SSTR2 involved in regulation of cell metabolism is unclear, the purpose of this study was to establish the CHO and HEK cell lines stably transfected with human SSTR2 that could be used as a model to study pharmacological properties of SSTR2 and to determine whether the
Somatostatin (SS) is known to have an antiproliferative effect on cell growth via somatostatin receptors (SSTR). The purpose of this study was to transfect cell lines with human SSTR2 and determine the subsequent effect on cell growth in response to SSTR agonist. Heterologous Chinese hamster ovary (CHO-K1) and human embryonic kidney 293 (HEK) cells were transfected with SSTR2 cDNA using lipofectin. Stable transformants were selected by G418 and confirmed by 125I-SS binding and RT–PCR. Binding studies were performed in the presence of 1006 to 10012 M SS-14, SS28, SS analogue RC-160, SSTR2 agonist NC-9-74, and SSTR5 agonist DC-37-39. Cell growth was determined by counting cell numbers after 48 hr incubation in the presence of 1006 to 10012 M SSTR2 agonist NC-9-74. Binding of 125I-SS-14 to transfected CHO and transfected HEK293 cells showed that the cells had high affinity for SS-14, SS-28, NC-9-74, and RC-160 but low affinity for DC-37-39. Incubation with 1006 to 10012 M NC-9-74, showed that 1 nM to 1 mM NC-9-74 significantly inhibited transfected HEK293 cell growth but did not affect growth on transfected CHO cells (n Å 4 for each dose, P õ 0.01). The two cell lines transfected with the human SSTR2 showed similar high affinity for SS-14, SS-28, RC-160, and SSTR2 agonist but not SSTR5 agonist. The SSTR2 agonist NC-9-74 significantly inhibited transfected HEK293 cell growth but not CHO cells. These data suggest that activation of SSTR2 was more efficiently coupled to the signal transduction pathway of antiproliferation in the transfected HEK293 cells. q 1997 Academic Press
INTRODUCTION
Somatostatin is well known to be a growth inhibitory hormone widely distributed throughout central and peripheral tissues [1–3]. It inhibits the release of many hormones, such as growth hormone, insulin, glucagon, gastrin, secretin, and vasoactive intestinal peptide and 1 To whom correspondence should be addressed at Department of Surgery, Baylor College of Medicine, 6550 Fannin, Ste. 447, Houston, Texas 77030. Fax: 713-793-1335. E-mail: [email protected].
0022-4804/97 $25.00 Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.
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transfection would result in an inhibition of cell growth in response to receptor-specific agonists. METHODS AND MATERIALS Construction of hSSTR2 expression vector and development of stable cell lines. Full-length cDNA encoding human somatostatin receptor type 2 (14) was subcloned into pcDNA3 vector (InVitrogen, San Diego, CA ) at EcoRI and XbaI sites. Human embryonic kidney 293 (HEK) cells were routinely cultured in Dulbecco’s modified Eagle medium:nutrient mixture F-12 (D-MEM/F-12) (GIBCO, Life Technologies, Inc., NY) supplemented with 10% fetal bovine serum (FBS) (GIBCO) at 377C in a humidified atmosphere of 5% CO2 . Chinese hamster ovary K1 (CHO) cells were cultured in HAM medium supplemented with 10% FBS (GIBCO) at 377C in a humidified atmosphere of 5% CO2 . For transfections of SSTR2, HEK and CHO cells were grown to 60–70% confluence in the 75-cm2 culture flask and transfected with 2 mg of expression vector SSTR2–pcDNA3 using lipofectin reagent (GIBCO-BRL) for 24 hr in serum-free medium. Cells were then returned to 10% FBS medium with 700 mg/ml G418 (GIBCO). G418-resistant colonies of HEK and CHO cells were selected by G418 and plated into 96-well plates to obtain a stable clone of single cell. The single-cell clones of both HEK and CHO cells were screened for somatostatin type 2 receptor by binding of 125I-somatostatin-14 (Amersham, Arlington Heights, IL). One of the stable clones from HEK and CHO cells was used to perform the studies of receptor binding and cell growth. Detection of SSTR2 from stable cells by RT–PCR. Total RNA were isolated from stable HEK and CHO cells using an RNAgents total RNA isolation system (Promega, Madison, WI). First-strand cDNA was synthesized using 1 mg of total RNA by reverse-transcription reaction (RT) (Promega, Madison, WI). First-strand cDNA was amplified by polymerase chain reaction (PCR) in a programmable thermal controller (M. J. Research, Inc., Watertown, MA) using the following specific primers: hSSTR2-1 (5* to 3*), GCA GCC ATG GAC ATG GCG GA (hSSTR2 nucleotides 77–96); hSSTR2-2 (5* to 3*), CCA AGC AGT TCA GAT ACT (hSSTR2 nucleotides 1184–1201); hSSTR2-3 (5* to 3 *): CAC ACA GCC ATG GTG ATC AT (SSTR2 nucleotides 563–582). Ten microliters of the amplified cDNA products from HEK and CHO cells was electrophoresed through 1% agarose gel and sequenced by the 310 Genetic Analyzer (Perkin Elmer, Foster, CA). Binding of 125I-somatostatin-14 to cells expressing SSTR2. Stable SSTR2–HEK-1 and SSTR–CHO-3 Cells grown to 80–90% confluence in a flask were plated onto 24-well plates and cultured for 2– 3 days in 1 ml of D-MEM/F-12 medium or HAM medium with 10% FBS. Binding experiments were performed in binding buffer containing 20 mM Hepes, pH 7.4, 0.1% bacitracin, 0.2% bovine serum albumin (BSA) in the presence of 125I-somatostatin-14 (20000 cpm/ well, Amersham) with or without somatostatin analogues. After incubation for 60 min at room temperature, cells were subsequently washed two times with 2 ml cold PBS and hydrolyzed with 1 ml of 0.1 M NaOH. Bound radioactivity was determined in a gamma counter (TracorAnalytic, Brandon, FL). All binding data were obtained from experiments repeated three to five times under the same conditions. Somatostatin-14, somatostatin-28, and somatostatin cyclic octapeptide analogue RC-160 were provided by Bachem Inc. (Torrance, CA). SSTR2 agonist NC 9-74 (NC-8-12) and SSTR5 agonist DC-37-39 were provided by D. H. Coy (Tulane University, New Orleans, LA). Control experiments with nontransfected cells and cells transfected only with pcDNA vector were performed under the same binding conditions. Cell growth study on transfected cells. The stable SSTR2–HEK1 and SSTR2–CHO-3 cells were plated on six-well dishes and cultured in the D-MEM/F-12 or HAM medium, both containing 10% FBS for 48 hr at 377C with or without 1006 to 10012M SSTR2 agonist NC 9-74. Cell growth was measured by counting the numbers of cells after the addition of 1 ml trypsin–EDTA to each well. Each individual experiment was measured in triplicate and repeated at least three times. SSTR2–HEK-1 and SSTR2–CHO-3 were cultured in the medium containing 10% FBS without NC-9-74 as controls. Non-
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FIG. 1. Detection of SSTR2 by RT–PCR from the cells. Ten milliliters of amplified cDNA fragments from stable SSTR2–HEK-1 and SSTR2–CHO-3 cells was eletrophoresed through a 1% agarose gel. Lane 1 was DNA standard. Lanes 2 and 3 were cDNA from SSTR2– HEK-1 cells. Lanes 4 and 5 were cDNA from SSTR2–CHO-3 cells. The 1128-bp cDNA products (lanes 2 and 4) using primers hSSTR21 and hSSTR2-2 encoded the full length of hSSTR 2. The 506-bp cDNA products (lanes 3 and 5) using primers hSSTR-1 and hSSTR23 encoded the partial length of hSSTR. The sequences of the cDNA products showed that they were hSSTR2.
transfected cell were incubated with NC-9-74 under the same conditions as for controls. Student’s t test was used in this study. Statistical analysis was performed by using Student’s t test. A value P õ 0.05 was considered significant.
RESULTS
The single-cell clones were screened for somatostatin receptor type 2 receptor by binding of 125I-somatostatin14. Seven of 12 HEK single-cell lines and 9 of 15 CHO single-cell lines showed high affinity to 125I-somatostatin-14, indicating these single-cell lines highly expressing somatostatin receptor type 2. Two stable cell lines, SSTR2–HEK-1 and SSTR2–CHO-3, were chosen to study RT–PCR, receptor binding, and cell growth. RT–PCR. RT–PCR was used to detect hSSTR2 in the transfected cells. Ten microliters of amplified cDNA fragments from stable SSTR2–HEK-1 and SSTR2– CHO-3 cells was electrophoresed through a 1% agarose gel, and 1128 base pairs (full length of hSSTR2) and 506 base pairs (partial length of hSSTR2) were obtained (Fig. 1). Sequencing of the cDNA products confirmed that they were hSSTR2. Receptor binding. SSTR2–HEK-1 cells were used to determine the binding affinity to somatostatin receptor agonism. The binding of 125I-somatostatin-14 to SSTR2–HEK-1 cells were displaced by somatostatin14, somatostatin-28, somatostatin analogue RC-160, SSTR2 agonist NC-9-74, and SSTR5 agonist DC-37-39 (Fig. 2). The binding of SSTR2–HEK-1 to 125I-somatostatin-14 was competitive with somatostatin-14 (IC50 Å 2 nM), somatostatin-28 (IC50 Å 2 nM), RC-160 (IC50 Å 4 nM), NC-9-74 (IC50 Å 6 nM), but not by DC-37-39 (IC50 is ú100 nM)(Table 1). A similar binding study was performed on SSTR2–CHO-3 (Fig 3). The binding of SSTR2–CHO-3 to 125I-somatostatin-14 was competitive with somatostatin-14, somatostatin-28, RC-160,
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FIG. 2. 125I-somatostatin-14 bound to stable HEK293 cells. Binding of 125I-somatostatin-14 (20,000 cpm/well) to stable SSTR2–HEK1 cells was measured in the binding buffer containing 20 mM Hepes, pH 7.4, 0.2% BSA in the presence of increasing concentrations of somatostain-14 (l), somatostatin-28 (j), somatostatin analogue RC160 (l), SSTR2 agonist NC-9-74 (m), and SSTR5 agonist DC 37-39 (∗). Cells were incubated for 60 min at room temperature. Each point represents the mean of three to five experiments. Maximal binding of each agonist represented the percentage of controls. Controls were 100% of 125I-somatostatin-14 binding to the cells without incubation with somatostatin analogues. Nontransfected cells and cells only transfected with pcDNA3 vector showed no binding to 125I-somatostatin-14.
and NC-9-74, but not by DC-37-39 and their IC50 were 0.9, 3, 3, 4, and ú100 nM, respectively (Table 1). The binding results suggest that both stable HEK and CHO cells highly expressed human somatostatin receptor type 2. Nontransfected HEK and CHO cells, and cells transfected only with pcDNA3 vector showed no binding to 125I-somatostatin-14. Cell growth. The effect of SSTR2 agonist NC-9-74 on the cell growth of transfected cells was studied. Cell proliferation of SSTR2–HEK-1 and SSTR2–CHO-3 cells was induced by D-MEM/F-12 or HAM medium contained 10% fetal bovine serum. Controls were the SSTR2–HEK-1 and SSTR2–CHO-3 cells without incubation with NC-9-74. The control cells represented 100% of cell growth. Incubation of cells for 48 hr with a concentration of 1 nM to 1 mM NC-9-74 inhibited SSTR2–HEK-1 growth compared to the controls (Fig. 4). A concentration of 1 nM inhibited SSTR2–HEK-1 cell proliferation with 62.2 { 17.0% (n Å 4, P õ 0.01)
FIG. 3. 125I-somatostatin-14 bound to stable CHO cells. Binding of 125I-somatostatin-14 (20,000 cpm/well) to stable SSTR2–CHO-3 cells was measured in the binding buffer containing 20 mM Hepes, pH 7.4, 0.2% BSA in the presence of increasing concentrations of somatostain-14 (l), somatostatin-28 (j), somatostatin analogue RC160 (l), SSTR2 agonist NC-9-74 (m), and SSTR5 agonist DC-37-39 (∗). Cells were incubated for 60 min at room temperature. Each point represents the mean of three to five experiments. Maximal binding of each agonist represented the percentage of controls. Controls were 100% of 125I-somatostatin-14 binding to the cells without incubation with somatostatin analogues. Nontransfected cells and cells only transfected with pcDNA3 vector showed no binding to 125I-somatostatin-14.
of control and maximal inhibition was 49.9 { 16.1% (n Å 4, P õ 0.01) of control using 1 mM NC-9-74. In contrast, NC-9-74 had no obvious inhibition of growth of SSTR2–CHO-3 cells. NC-9-74 had no effect on growth of nontransfected cells. DISCUSSION
The recent molecular cloning of five somatostatin receptors has contributed to the understanding of the pharmacological properties and mechanisms of signal transduction of these receptors in the transfected cell lines. In present study, HEK and CHO cells were stably transfected with hSSTR2 to determine receptor binding affinity and to determine whether the transfection would result in an inhibition of cell growth in response to receptor-specific agonists. The HEK and CHO cells were chosen because neither express SSTR2. Infusion of SSTR2 agonist into the isolated perfused human pancreas resulted in a significant inhibition of insulin
TABLE 1 125
Comparison of the Binding Affinity of I-Somatostatin-14 to Stable SSTR2–HEK1 and SSTR2–CHO-3 Cells and the Effect of Transfected Cell Growth in Response to SSTR2 Agonist NC-9-74 Binding affinity (IC50 nM)
HEK CHO
AID
Cell growth
SS-14
SS-28
NC-9-74
RC-160
DC-37-39
1 nM–1 mM of NC-9-74
2 0.9
2 3
4 3
6 4
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FIG. 4. The stable SSTR2–HEK-1 (h) and SSTR2–CHO-3 (l) cells were plated on six-well dishes and were cultured in the D-MEM/ F-12 or HAM medium, both containing 10% FBS for 48 hr at 377C with or without 1006 to 10012 M SSTR2 agonist NC-9-74. Cell growth was measured by counting cell numbers after addition of 1 ml trypsin–EDTA to each well. Each individual experiment was measured in triplicate and repeated at least three times. Controls were the stable SSTR2–HEK-1 and SSTR2–CHO-3 cells and were measured as 100% of growth without incubation with NC-9-74. NC-9-74 showed no effect on nontransfected cells.
and C-peptide secretion, suggesting that the inhibitory effect of somatostatin on b-cell secretion is mediated through the type 2 receptor within the human islet (26). We therefore chose the hSSTR2 for this study in order to characterize its pharmacological properties and its ability to inhibit cell growth. Both CHO and HEK cells were stably transfected with hSSTR2. 125I-somatostatin-14 was used to select the single cell lines which had a high affinity for 125Isomatostatin-14. In order to confirm the presence of SSTR2 in HEK293 and CHO cells, RT–PCR was used to detect SSTR2. The two expected cDNA fragments were obtained which encoding 1128 base pairs of the full-length SSTR2 and 506 base pairs of the partiallength SSTR2. DNA sequencing confirmed that these two amplified cDNA fragments were hSSTR2 cDNA. These data suggest that the two stably transfected cell lines express the human somatostatin receptor type-2. SSTRs are seven transmembrane domain G-proteincoupled receptors and coupled to multiple second messenger systems via pertussis toxin-sensitive or -insensitive guanine G-protein. SSTR1-5 are coupled to adenylyl cyclase to inhibit forskolin-stimulated cAMP accumulation [16, 27–31]. Activation of SSTR2 receptor in transfected cells was able to increase calcium mobilization via phospholipase C [32, 33]. In rat AR42J cells, SSTR2 mediated phospholipase C-independent Ca2/ mobilization by opening cell-surface calcium channels [34]. Somatostatin and its analogues has been used to inhibit growth of tumor cells derived from pancreas [35], breast [36, 37], colon [38], thyroid [39], prostate [40], lung [41], and pituitary and brain [42]. The inhibition of tumor cell growth by somatostatin was related to the activity of phosphotyrosine phosphatase [36, 43]. Recently, it have been reported that in trans-
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fected hSSTR2 cells such as COS-7 and NIH 3T3, somatostatin analogues RC-160 and SMS 201-995 inhibited transfected cell growth by stimulating tyrosine phosphatase activity [44]. The pharmacological characteristics of somatostatin receptor type 2 for various of somatostatin analogues have been studied in different species using labeled somatostatin and its analogues. SSTR2 cloned from mouse, rat, and human tissues exhibited high and similar affinity for somatostatin-14 and somatostatin-28 in transfected CHO-G44, CHO-K1, and COS1 cells. [14, 23, 25]. The 125I-MK-678 (a somatostatin hexapeptide analogue) binding to mouse SSTR2 was inhibited by somatostatin and its analogue RC 160 in the transfected CHO cells [45, 46]. hSSTR2 bound with labeled somatostatin-28 was inhibited by somatostatin analogue RC-160 and SMS 201-995 (Octreotide) in transfected CHO-K1 cells [25]. RC-160 and SMS 201-995 were also used in the studies of the other SSTR subtypes. RC-160 had high binding affinity for SSTR3-5, but not SSTR1 [25]. SMS 201-995 had high binding affinity for SSTR3 and SSTR5, but not for SSTR1 and SSTR4 [25, 46]. In this study, the receptor binding affinity of transfected HEK and CHO cells was studied using somatostatin-14, somatostatin-28, RC-160, and NC-9-74 which is specific for SSTR2, and DC-37-39 which is specific for SSTR5 [25, 47]. The two stable transfected cell lines showed high affinity for somatostatin-14, somatostatin-28, somatostatin analogue RC-160, and SSTR2specific agonist NC-9-74. SSTR2 had binding affinity for NC-9-74 similar to that of somatostatin-14 and somatostatin-28, confirming that NC-9-74 was a specific agonist for SSTR2. The close IC50 values of two stable cell lines binding the somatostatin analogues reflected that there is no difference in binding affinity of the two types of transfected cells. On the other hand, DC-3739, a selective SSTR5 agonist, bound both HEK and CHO with very low affinity, indicating that there is a pharmacological difference between SSTR2 and SSTR5. These data demonstrated that the transfected CHO and HEK cells have a high affinity for the SSTR2 agonist but not for the SSTR5 agonist. In the cell growth study, we found for first time that SSTR2-specific agonist NC-9-74 inhibited growth of HEK cells stably transfected with human SSTR2 in the medium containing 10% fetal bovine serum, but not stably transfected CHO cells. These data imply that hSSTR2 has an antiproliferative role and that HEK cells have the intracellular environment for allowing SSTR2 to mediate cell antiproliferation, whereas the CHO cells do not. The latter could be explained by the lack of the proper G-protein (Gia1) in the CHO cells, since RC 160 was able to inhibit CHO cell proliferation when these cells were transfected with mouse SSTR2 with G-protein Gia1 [48]. In contrast, somatostatin failed to increase tyrosine phosphatase activity in CHO-K1 transfected with rat SSTR2 [49]. In conclusion, the results of the present study demonstrate that human somatostatin receptor type 2 can be
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transfected into HEK and CHO cells and that these stable cell lines show binding similar to somatostatin analogues. SSTR2-specific agonist NC-9-74 significantly inhibited growth of transfected HEK cells but not CHO cells, suggesting that activation of SSTR2 was more efficiently coupled to the signal transduction pathway of antiproliferation in the transfected HEK cells than in the CHO cells. This study confirms the specificity of the SSTR2 agonist for the human SSTR2 receptor. Further studies are necessary to determine the signal transduction pathway of antiproliferation in the transfected SSTR cells. ACKNOWLEDGMENT This work was supported by NIH Grant R29DK46441-01.
REFERENCES 1. Reichlin, S. Somatostatin. N. Engl. J. Med.309: 1495, 1983. 2. Reisine, T., and Bell, G. I. Molecular properties of somatostatin receptors. Neuroscience 67: 777, 1995. 3. Patel, Y. C., Greenwood, M. T., Panetta, R., Demchyshyn, L., Niznik, H., and Srikant, C. B. Mini review the somatostatin receptor. Fam. Life Sci. 57: 1249, 1995. 4. Bloom, S., Mortimer, C., Thorner, M., Besser, G., Hall, R., et al. Inhibition of gastrin and gastric acid secretion by growth hormone release-inhibitory hormone. Lancet 2: 1106, 1974. 5. Tannenbaum, G., Ling, N., and Brazeau, P. SRIF 28 is longer acting and more selective than SRIF on pituitary and pancreatic hormone release. Endocrinology 111: 101, 1982. 6. Gomez-pan, A., Reed, J., Albinus, M., Shaw, B., et al. Direct of gastric acid and pepsin secretion by growth-hormone releaseinhibiting hormone in rat. Lancet 1: 888, 1975. 7. Epelbaum, J. Somatostatin in the central nervous system: Physiology and pathological modification. Prog. Neurobiol. 27: 63, 1986. 8. Brazeau, P., Vale, W., Burgus, R., Ling, N., Rivier, J., and Guillemin, R. Hypothalamic polypeptide that inhibits the secretion of immunoreactive pituitary growth hormone. Science 129: 77, 1973. 9. Pradayrol, L., Jornvall, H., Mutt, V., et al. N-terminally extended somatostatin: The primary structure of somatostatin28. FEBS Lett. 109: 55, 1980. 10. Schally, A. V. Oncological application of somatostatin analogues. Cancer Res. 48: 6977, 1988. 11. Vanetti, M., Kouba, M., Wang, X., Vogt, G., and Hoellt, V. Cloning and expression of a novel mouse somatostatin receptor (SSTR2B). FEBS Lett. 311: 290, 1992. 12. Patel, Y. C., Greenwood, M., Kent, B., Panetta, R., and Srikant, C. B. Multiple gene transcripts of the somatostatin receptor SSTR2: Tissue selective distribution and cAMP regulation. Biochem. Biophys. Res. Commun. 192: 288, 1993. 13. Taylor, J. E., Theveniau, M. A., Bashirzadeh, R., Resine, T., and Eden, P. A. Detection of somatostatin receptor subtype 2 (SSTR2) in established tumors and tumor cell lines: Evidence for SSTR2 heterogeneity. Peptides 15: 1229, 1994. 14. Yamada, Y., Post, S. R., Wang, K., et al. Cloning and functional characterization of a family of human and mouse somatostatin receptors expressed in brain, gastrointestinal tract, and kidney. Proc. Natl. Acad. Sci. USA 89: 251, 1992. 15. Yamada, Y., Reisine, T., Law, S. F., et al. Somatostatin receptors, an expanding Gene family: Cloning and human SSTR3, a protein coupled to adenylyl cyclase. Mol. Endocrinol. 6: 2136, 1992.
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16. Yamada, Y., Kagimoto, S., Kubota, A., et al. Cloning, functional expression and pharmacological characterization of a fourth (hSSTR4) and a fifth (hSSTR5) human somatostatin receptor subtype. Biochem. Biophys. Res. Commun. 195: 844, 1993. 17. Rohrer, L., Raulf, F., Bruns, C., et al. Cloning and characterization of a fourth human somatostatin receptor. Proc. Natl. Acad. Sci. 90: 4196, 1993. 18. Panetta, R., Greenwood, M. T., Warszynska, A., et al. Molecular cloning, functional characterization and chromosomal localization of a human somatostatin receptor (somatostatin receptor type 5) with preferential affinity with somatostatin-28. Mol. Pharmacol. 45: 417, 1994. 19. Breder, C. D., Yamada, Y., Yasuda, K., Seino, S., Saper, C., and Bell, G. I. Differential expression of SRIF receptor subtypes in brain. J. Neurosci. 12: 3920, 1992. 20. Bruno, J. F., Xu, Y., Song, J., and Berelowitz, M. Tissue distribution of SRIF receptor subtype messenger ribonucleic acid in the rat. Endocrinology 133: 2561, 1993. 21. Kong, H., DePaoli, A. M., Breder, C. D., Yasuda, K., Bell, G. I., and Reisine, T. Differential expression of SRIF receptor subtypes SSTR1, SSTR2 and SSTR3 in adult rat brain, pituitary and adrenal gland. Analysis by RNA blotting and in situ hybridization. Neuroscience 59: 175, 1994. 22. Raulf, F., Perez, J., Hoyer, D., and Bruns, C. Differential expression of five somatostatin receptor subtypes, SSTR1-5, in the CNS and peripheral tissue. Digestion 55: 46, 1994. 23. Kluxen, F. W., Bruns, C., and Luebbert, H. Expression and cloning of a rat brain somatostatin receptor cDNA. Proc. Natl. Acad. Sci. 89: 4618, 1992. 24. O’Carroll, A. M., Lolait, S. J., Konig, M., and McMahan, L. C. Molecular cloning and expression of a pituitary somatostatin receptor with preferential affinity for somatostatin-28. Mol. Pharmacol. 42: 939, 1992. 25. Patel, Y. C., and Srikant, C. B. Subtype selectivity of peptide analogs for all five cloned human somatostatin receptors (hSSTR1-5). Endocinology 135(6): 2814, 1994. 26. Moldovan, S., Atiya, A., Adrian, T. E., Kleinman, R. M., et al. Somatostatin inhibits B-cell secretion via a subtype-2 somatostatin receptor in the isolated perfused human pancreas. J. Surg. Res. 59: 85, 1995. 27. Kaupmann, K., Bruns, C., Hoyer, D., Seuwen, K., and Luebbert, H. Distribution and second messenger coupling of four somatostatin receptor subtypes expressed in brain. FEBS Lett. 331: 53, 1993. 28. Strnad, J., Eppler, C. M., Corbett, M., and Hadcork, J. R. The rat SSTR2 somatostatin rceptor subtype is coupled to inhibition of cyclic AMP accumulation. Biochem. Biophys. Res. Commun. 191: 968, 1993. 29. Yasuda, K., Rens-Domiano, S., Breder, C.D., et al. Cloning of a novel somatostatin receptor, sstr3, coupled to adenylyl cyclase. J. Biol. Chem. 267: 20422, 1992. 30. Raynor, K., O’Carroll, A. M., Kong, H. Y., et al. Characterization of cloned somatostatin receptors SSTR4 and SSTR5. Mol. Pharmacol. 44: 385, 1993. 31. Patel, Y. C., Greenwood, M. T., Warszynska, A., Panetta, R., and Srikant, C. B. All five cloned human somatostatin rceptors (hSSTR1-5) are functionally coupled to adenylyl cyclase. Biochem. Biophys. Res. Commun. 198: 605, 1994. 32. Tomura, H., Okajima, F., Akbar, M., Majid, M. A., Sho, K., and Kondo, Y. Transfected human somatostatin receptors type 2, SSTR2, not only inhibits adenylate cyclase but also stimulates phospholipase C and Ca2/ mobilization. Biochem. Biophys. Res. Commun. 200: 986, 1994. 33. Akbar, M., Okajima, F., Tomura, H., Majid, M. A., Yamada, Y., Seino, S., and Kondo, Y. Phospholipase C activation and Ca2/ mobilization by cloned human somatostatin receptor subtypes 1-5, in transfected COS-7 cells. FEBS Lett. 348: 192, 1994. 34. Taylor, J. E. Somatostatin (SSTR2) receptors mediate phospho-
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42.
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JOURNAL OF SURGICAL RESEARCH: VOL. 71, NO. 1, JULY 15, 1997 lipase C-independent Ca2/ mobilization in rat AR42J pancreas cells. Biochem. Biophys. Res. Commun. 214: 81, 1995. Liebow, C., Reilly, C., Serrano, M., and Schally, A. V. Somatostatin analogues inhibit growth of pancreatic cancer by stimulating tyrosine phosphatase. Proc. Natl. Acad. Sci. USA 86: 2003, 1989. Setyono-Han, B., Henkelman, M. S., Foekens, J. A., and Klijn J. G. M. Direct inhibitory effects of somatostatin (analogues) on the growth of human breast cancer cell lines. Cancer Res. 47: 1566, 1987. Scambia, G., Panici, P. B., Baiocchi, G., Perrone, L., Oacobelli, S., and Mancuso, S. Antiproliferative effects of somatostatin and the somatostatin analog SMS 201-995 on three human breast cancer cell lines. J. Cancer Res. Clin. Oncol. 114: 306, 1988. Qin, Y., Schally, A. V., and Willems, G. Somatostatin analogues RC-160 inhibits the growth of transplanted colon cancer in rats. Int. J. Cancer 47: 765, 1991. Tsuzaki, S., and Moses, A. C. Somatostatin inhibits deoxyribonucleic acid synthesis induced by both thyrotropin and insulinlike growth factor-1 in FRTL5 cells. Endocrinology 126: 3131, 1990. Schally, A. V., and Redding, R. W. Somatostatin analogs as adjuncts to agonist of luteinizing hormone-releasing hormone in the treatment of experimental prostate cancer. Proc. Natl. Acad. Sci. USA 84: 7275, 1987. Taylor, J. E., Moreau J. P., Baptiste, L., and Moody, T. W. Octapeptide analogues of somatostatin inhibit the clonal growth and vasoactive intestinal peptide-stimulated cyclic AMP formation in human small cell lung cancer cells. Peptides 12: 839, 1990. Lamberts, S. W. J., Krenning, E. P., and Reubi, J. C. The role of somatostatin and its analogs in the diagnosis and treatment of tumors. Endocrinol. Rev. 12: 450, 1991.
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43. Lee, M. T., Liebow, C., Kamer, A. R., and Schally, A. V. Effects of epidermal growth factor and analogues of luteinizing hormone-releasing hormone and somatostatin on phosphorylation and dephosphorylation of tyrosine residues of specific protein substrates in various tumors. Proc. Natl. Acad. Sci. USA 88: 1656, 1991. 44. Buscail, L., Delesque, N., Esteve, J. P., Saint-Laurent, N., et al. Stimulation of tyosine phosphatase and inhibition of cell proliferation by somatostatin analogues: Mediation by human somatostatin receptor subtypes SSTR1 and SSTR2. Proc. Natl. Acad. Sci. USA 91: 2315, 1994. 45. Rens-Domiano, S., Law, S. F., Yamada, Y., Seino, S., Bel, G. I., and Reisine, T. Pharmacological properties of two cloned SRIF receptors. Mol. Pharmacol. 42: 28, 1992. 46. Raynor, K., Murphy, W. A., Coy, D. H., et al. Cloned somatostatin receptors: Identification of subtype-selective peptides and demonstration of high affinity binding of linear peptides. Mol. Pharmacol. 43: 838, 1993. 47. Rossowski, W. J., and Coy, D. H. Specific inhibition of rat pancreatic insulin or glucagon release by receptor-selective somatostatin analogs. Biochem. Biophys. Res. Commun. 205(1): 341, 1994. 48. Buscail, L., Esteve, J. P., Saint-Laurent, N., Bertrand, V., Reisine, T., O’Carroll, A. M., Bell, G. I., Schally, A., Vaysse, N., and Susini, C. Inhibition of cell proliferation by the somatostatin analogue RC-160 is mediated by SSTR2 and SSTR5 somatostatin receptor subtypes through different mechanisms. Proc. Natl. Acad. Sci. USA 92: 1580, 1995. 49. Florio, T., Rim, C., Hershberger, R. E., Loda, M., and Stork, P. J. The somatostatin receptor SSTR1 is coupled to phosphotyrosine phosphatase activity in CHO-K1 cells. Mol. Endocrinol. 8(10): 1289, 1994.
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Activation of Human Somatostatin Receptor Type 2 Causes Inhibition of Cell Growth in Transfected HEK293 but Not in Transfected CHO Cells J. Ren,* G. Bell,† D. H. Coy,‡ and F. C. Brunicardi*,1 *Department of Surgery, Baylor College of Medicine, Houston, Texas 77030; †Howard Hughes Medical Institute, University of Chicago, Chicago, Illinois 60637; and ‡Tulane University, New Orleans, Louisiana 70118 Presented at the Annual Meeting of the Association for Academic Surgery, Chicago, Illinois, November 13–16, 1996
acts via activation of somatostatin receptors [4–8]. There are two naturally occurring forms of bioactive somatostatin, the 14-amino-acid-containing somatostatin (SS-14) and its N-terminally extended precursor somatostatin-28 (SS-28) [8, 9]. The antiproliferative effect of somatostatin has been studied in vitro and in vivo, and this peptide may participate in the regulation of cell growth both in normal and tumor cells [10]. Recently, a family of somatostatin receptors (SSTR) has been cloned and referred to as SSTR1-5 [2, 3]. SSTRs are seven transmembrane domain guanine nucleotide binding protein (G-protein)-coupled receptors and are coupled to multiple second messenger systems. Mouse, human and rat SSTR 2 generate two separate isoforms, a long (SSTR2A) and a short (SSTR2B) variant through alternate mRNA splicing at the C-terminal portion [11–13]. mRNAs for the five receptor subtypes are expressed in a tissue- and species-specific manner. Human SSTR1-5 mRNA is expressed in the brain, jejunum, stomach, pancreatic islets, and lung [14–18]. mRNA expression of both rat SSTR1-5 and mouse SSTR1-3 are found in the brain [14, 19–23]. The rat stomach expresses the four subtypes except SSTR5, while the liver and pancreas express only SSTR3 and SSTR2, respectively [20]. The distribution of somatostatin receptor type 2B is detected in brain and pituitary of rat and mouse, human pituitary tumors, and a human small cell lung cancer cell line [11–13]. The abundance of SSTR2B is higher than that of SSTR2A in mouse brain [11]. SSTR1-4 show similar high affinity with somatostatin-14 and somatostatin-28 [14–17]. On the other hand, rSSTR5 and hSSTR5 exhibit the higher affinity for somatostatin-28 than somatostatin-14 [16, 18, 24, 25]. We previously demonstrated that insulin release from human islets was inhibited by SSTR2 agonist and that the human pancreatic b cell was primarily occupied by SSTR2 [26]. Since mechanism of human SSTR2 involved in regulation of cell metabolism is unclear, the purpose of this study was to establish the CHO and HEK cell lines stably transfected with human SSTR2 that could be used as a model to study pharmacological properties of SSTR2 and to determine whether the
Somatostatin (SS) is known to have an antiproliferative effect on cell growth via somatostatin receptors (SSTR). The purpose of this study was to transfect cell lines with human SSTR2 and determine the subsequent effect on cell growth in response to SSTR agonist. Heterologous Chinese hamster ovary (CHO-K1) and human embryonic kidney 293 (HEK) cells were transfected with SSTR2 cDNA using lipofectin. Stable transformants were selected by G418 and confirmed by 125I-SS binding and RT–PCR. Binding studies were performed in the presence of 1006 to 10012 M SS-14, SS28, SS analogue RC-160, SSTR2 agonist NC-9-74, and SSTR5 agonist DC-37-39. Cell growth was determined by counting cell numbers after 48 hr incubation in the presence of 1006 to 10012 M SSTR2 agonist NC-9-74. Binding of 125I-SS-14 to transfected CHO and transfected HEK293 cells showed that the cells had high affinity for SS-14, SS-28, NC-9-74, and RC-160 but low affinity for DC-37-39. Incubation with 1006 to 10012 M NC-9-74, showed that 1 nM to 1 mM NC-9-74 significantly inhibited transfected HEK293 cell growth but did not affect growth on transfected CHO cells (n Å 4 for each dose, P õ 0.01). The two cell lines transfected with the human SSTR2 showed similar high affinity for SS-14, SS-28, RC-160, and SSTR2 agonist but not SSTR5 agonist. The SSTR2 agonist NC-9-74 significantly inhibited transfected HEK293 cell growth but not CHO cells. These data suggest that activation of SSTR2 was more efficiently coupled to the signal transduction pathway of antiproliferation in the transfected HEK293 cells. q 1997 Academic Press
INTRODUCTION
Somatostatin is well known to be a growth inhibitory hormone widely distributed throughout central and peripheral tissues [1–3]. It inhibits the release of many hormones, such as growth hormone, insulin, glucagon, gastrin, secretin, and vasoactive intestinal peptide and 1 To whom correspondence should be addressed at Department of Surgery, Baylor College of Medicine, 6550 Fannin, Ste. 447, Houston, Texas 77030. Fax: 713-793-1335. E-mail: [email protected].
0022-4804/97 $25.00 Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.
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transfection would result in an inhibition of cell growth in response to receptor-specific agonists. METHODS AND MATERIALS Construction of hSSTR2 expression vector and development of stable cell lines. Full-length cDNA encoding human somatostatin receptor type 2 (14) was subcloned into pcDNA3 vector (InVitrogen, San Diego, CA ) at EcoRI and XbaI sites. Human embryonic kidney 293 (HEK) cells were routinely cultured in Dulbecco’s modified Eagle medium:nutrient mixture F-12 (D-MEM/F-12) (GIBCO, Life Technologies, Inc., NY) supplemented with 10% fetal bovine serum (FBS) (GIBCO) at 377C in a humidified atmosphere of 5% CO2 . Chinese hamster ovary K1 (CHO) cells were cultured in HAM medium supplemented with 10% FBS (GIBCO) at 377C in a humidified atmosphere of 5% CO2 . For transfections of SSTR2, HEK and CHO cells were grown to 60–70% confluence in the 75-cm2 culture flask and transfected with 2 mg of expression vector SSTR2–pcDNA3 using lipofectin reagent (GIBCO-BRL) for 24 hr in serum-free medium. Cells were then returned to 10% FBS medium with 700 mg/ml G418 (GIBCO). G418-resistant colonies of HEK and CHO cells were selected by G418 and plated into 96-well plates to obtain a stable clone of single cell. The single-cell clones of both HEK and CHO cells were screened for somatostatin type 2 receptor by binding of 125I-somatostatin-14 (Amersham, Arlington Heights, IL). One of the stable clones from HEK and CHO cells was used to perform the studies of receptor binding and cell growth. Detection of SSTR2 from stable cells by RT–PCR. Total RNA were isolated from stable HEK and CHO cells using an RNAgents total RNA isolation system (Promega, Madison, WI). First-strand cDNA was synthesized using 1 mg of total RNA by reverse-transcription reaction (RT) (Promega, Madison, WI). First-strand cDNA was amplified by polymerase chain reaction (PCR) in a programmable thermal controller (M. J. Research, Inc., Watertown, MA) using the following specific primers: hSSTR2-1 (5* to 3*), GCA GCC ATG GAC ATG GCG GA (hSSTR2 nucleotides 77–96); hSSTR2-2 (5* to 3*), CCA AGC AGT TCA GAT ACT (hSSTR2 nucleotides 1184–1201); hSSTR2-3 (5* to 3 *): CAC ACA GCC ATG GTG ATC AT (SSTR2 nucleotides 563–582). Ten microliters of the amplified cDNA products from HEK and CHO cells was electrophoresed through 1% agarose gel and sequenced by the 310 Genetic Analyzer (Perkin Elmer, Foster, CA). Binding of 125I-somatostatin-14 to cells expressing SSTR2. Stable SSTR2–HEK-1 and SSTR–CHO-3 Cells grown to 80–90% confluence in a flask were plated onto 24-well plates and cultured for 2– 3 days in 1 ml of D-MEM/F-12 medium or HAM medium with 10% FBS. Binding experiments were performed in binding buffer containing 20 mM Hepes, pH 7.4, 0.1% bacitracin, 0.2% bovine serum albumin (BSA) in the presence of 125I-somatostatin-14 (20000 cpm/ well, Amersham) with or without somatostatin analogues. After incubation for 60 min at room temperature, cells were subsequently washed two times with 2 ml cold PBS and hydrolyzed with 1 ml of 0.1 M NaOH. Bound radioactivity was determined in a gamma counter (TracorAnalytic, Brandon, FL). All binding data were obtained from experiments repeated three to five times under the same conditions. Somatostatin-14, somatostatin-28, and somatostatin cyclic octapeptide analogue RC-160 were provided by Bachem Inc. (Torrance, CA). SSTR2 agonist NC 9-74 (NC-8-12) and SSTR5 agonist DC-37-39 were provided by D. H. Coy (Tulane University, New Orleans, LA). Control experiments with nontransfected cells and cells transfected only with pcDNA vector were performed under the same binding conditions. Cell growth study on transfected cells. The stable SSTR2–HEK1 and SSTR2–CHO-3 cells were plated on six-well dishes and cultured in the D-MEM/F-12 or HAM medium, both containing 10% FBS for 48 hr at 377C with or without 1006 to 10012M SSTR2 agonist NC 9-74. Cell growth was measured by counting the numbers of cells after the addition of 1 ml trypsin–EDTA to each well. Each individual experiment was measured in triplicate and repeated at least three times. SSTR2–HEK-1 and SSTR2–CHO-3 were cultured in the medium containing 10% FBS without NC-9-74 as controls. Non-
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FIG. 1. Detection of SSTR2 by RT–PCR from the cells. Ten milliliters of amplified cDNA fragments from stable SSTR2–HEK-1 and SSTR2–CHO-3 cells was eletrophoresed through a 1% agarose gel. Lane 1 was DNA standard. Lanes 2 and 3 were cDNA from SSTR2– HEK-1 cells. Lanes 4 and 5 were cDNA from SSTR2–CHO-3 cells. The 1128-bp cDNA products (lanes 2 and 4) using primers hSSTR21 and hSSTR2-2 encoded the full length of hSSTR 2. The 506-bp cDNA products (lanes 3 and 5) using primers hSSTR-1 and hSSTR23 encoded the partial length of hSSTR. The sequences of the cDNA products showed that they were hSSTR2.
transfected cell were incubated with NC-9-74 under the same conditions as for controls. Student’s t test was used in this study. Statistical analysis was performed by using Student’s t test. A value P õ 0.05 was considered significant.
RESULTS
The single-cell clones were screened for somatostatin receptor type 2 receptor by binding of 125I-somatostatin14. Seven of 12 HEK single-cell lines and 9 of 15 CHO single-cell lines showed high affinity to 125I-somatostatin-14, indicating these single-cell lines highly expressing somatostatin receptor type 2. Two stable cell lines, SSTR2–HEK-1 and SSTR2–CHO-3, were chosen to study RT–PCR, receptor binding, and cell growth. RT–PCR. RT–PCR was used to detect hSSTR2 in the transfected cells. Ten microliters of amplified cDNA fragments from stable SSTR2–HEK-1 and SSTR2– CHO-3 cells was electrophoresed through a 1% agarose gel, and 1128 base pairs (full length of hSSTR2) and 506 base pairs (partial length of hSSTR2) were obtained (Fig. 1). Sequencing of the cDNA products confirmed that they were hSSTR2. Receptor binding. SSTR2–HEK-1 cells were used to determine the binding affinity to somatostatin receptor agonism. The binding of 125I-somatostatin-14 to SSTR2–HEK-1 cells were displaced by somatostatin14, somatostatin-28, somatostatin analogue RC-160, SSTR2 agonist NC-9-74, and SSTR5 agonist DC-37-39 (Fig. 2). The binding of SSTR2–HEK-1 to 125I-somatostatin-14 was competitive with somatostatin-14 (IC50 Å 2 nM), somatostatin-28 (IC50 Å 2 nM), RC-160 (IC50 Å 4 nM), NC-9-74 (IC50 Å 6 nM), but not by DC-37-39 (IC50 is ú100 nM)(Table 1). A similar binding study was performed on SSTR2–CHO-3 (Fig 3). The binding of SSTR2–CHO-3 to 125I-somatostatin-14 was competitive with somatostatin-14, somatostatin-28, RC-160,
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FIG. 2. 125I-somatostatin-14 bound to stable HEK293 cells. Binding of 125I-somatostatin-14 (20,000 cpm/well) to stable SSTR2–HEK1 cells was measured in the binding buffer containing 20 mM Hepes, pH 7.4, 0.2% BSA in the presence of increasing concentrations of somatostain-14 (l), somatostatin-28 (j), somatostatin analogue RC160 (l), SSTR2 agonist NC-9-74 (m), and SSTR5 agonist DC 37-39 (∗). Cells were incubated for 60 min at room temperature. Each point represents the mean of three to five experiments. Maximal binding of each agonist represented the percentage of controls. Controls were 100% of 125I-somatostatin-14 binding to the cells without incubation with somatostatin analogues. Nontransfected cells and cells only transfected with pcDNA3 vector showed no binding to 125I-somatostatin-14.
and NC-9-74, but not by DC-37-39 and their IC50 were 0.9, 3, 3, 4, and ú100 nM, respectively (Table 1). The binding results suggest that both stable HEK and CHO cells highly expressed human somatostatin receptor type 2. Nontransfected HEK and CHO cells, and cells transfected only with pcDNA3 vector showed no binding to 125I-somatostatin-14. Cell growth. The effect of SSTR2 agonist NC-9-74 on the cell growth of transfected cells was studied. Cell proliferation of SSTR2–HEK-1 and SSTR2–CHO-3 cells was induced by D-MEM/F-12 or HAM medium contained 10% fetal bovine serum. Controls were the SSTR2–HEK-1 and SSTR2–CHO-3 cells without incubation with NC-9-74. The control cells represented 100% of cell growth. Incubation of cells for 48 hr with a concentration of 1 nM to 1 mM NC-9-74 inhibited SSTR2–HEK-1 growth compared to the controls (Fig. 4). A concentration of 1 nM inhibited SSTR2–HEK-1 cell proliferation with 62.2 { 17.0% (n Å 4, P õ 0.01)
FIG. 3. 125I-somatostatin-14 bound to stable CHO cells. Binding of 125I-somatostatin-14 (20,000 cpm/well) to stable SSTR2–CHO-3 cells was measured in the binding buffer containing 20 mM Hepes, pH 7.4, 0.2% BSA in the presence of increasing concentrations of somatostain-14 (l), somatostatin-28 (j), somatostatin analogue RC160 (l), SSTR2 agonist NC-9-74 (m), and SSTR5 agonist DC-37-39 (∗). Cells were incubated for 60 min at room temperature. Each point represents the mean of three to five experiments. Maximal binding of each agonist represented the percentage of controls. Controls were 100% of 125I-somatostatin-14 binding to the cells without incubation with somatostatin analogues. Nontransfected cells and cells only transfected with pcDNA3 vector showed no binding to 125I-somatostatin-14.
of control and maximal inhibition was 49.9 { 16.1% (n Å 4, P õ 0.01) of control using 1 mM NC-9-74. In contrast, NC-9-74 had no obvious inhibition of growth of SSTR2–CHO-3 cells. NC-9-74 had no effect on growth of nontransfected cells. DISCUSSION
The recent molecular cloning of five somatostatin receptors has contributed to the understanding of the pharmacological properties and mechanisms of signal transduction of these receptors in the transfected cell lines. In present study, HEK and CHO cells were stably transfected with hSSTR2 to determine receptor binding affinity and to determine whether the transfection would result in an inhibition of cell growth in response to receptor-specific agonists. The HEK and CHO cells were chosen because neither express SSTR2. Infusion of SSTR2 agonist into the isolated perfused human pancreas resulted in a significant inhibition of insulin
TABLE 1 125
Comparison of the Binding Affinity of I-Somatostatin-14 to Stable SSTR2–HEK1 and SSTR2–CHO-3 Cells and the Effect of Transfected Cell Growth in Response to SSTR2 Agonist NC-9-74 Binding affinity (IC50 nM)
HEK CHO
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Cell growth
SS-14
SS-28
NC-9-74
RC-160
DC-37-39
1 nM–1 mM of NC-9-74
2 0.9
2 3
4 3
6 4
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FIG. 4. The stable SSTR2–HEK-1 (h) and SSTR2–CHO-3 (l) cells were plated on six-well dishes and were cultured in the D-MEM/ F-12 or HAM medium, both containing 10% FBS for 48 hr at 377C with or without 1006 to 10012 M SSTR2 agonist NC-9-74. Cell growth was measured by counting cell numbers after addition of 1 ml trypsin–EDTA to each well. Each individual experiment was measured in triplicate and repeated at least three times. Controls were the stable SSTR2–HEK-1 and SSTR2–CHO-3 cells and were measured as 100% of growth without incubation with NC-9-74. NC-9-74 showed no effect on nontransfected cells.
and C-peptide secretion, suggesting that the inhibitory effect of somatostatin on b-cell secretion is mediated through the type 2 receptor within the human islet (26). We therefore chose the hSSTR2 for this study in order to characterize its pharmacological properties and its ability to inhibit cell growth. Both CHO and HEK cells were stably transfected with hSSTR2. 125I-somatostatin-14 was used to select the single cell lines which had a high affinity for 125Isomatostatin-14. In order to confirm the presence of SSTR2 in HEK293 and CHO cells, RT–PCR was used to detect SSTR2. The two expected cDNA fragments were obtained which encoding 1128 base pairs of the full-length SSTR2 and 506 base pairs of the partiallength SSTR2. DNA sequencing confirmed that these two amplified cDNA fragments were hSSTR2 cDNA. These data suggest that the two stably transfected cell lines express the human somatostatin receptor type-2. SSTRs are seven transmembrane domain G-proteincoupled receptors and coupled to multiple second messenger systems via pertussis toxin-sensitive or -insensitive guanine G-protein. SSTR1-5 are coupled to adenylyl cyclase to inhibit forskolin-stimulated cAMP accumulation [16, 27–31]. Activation of SSTR2 receptor in transfected cells was able to increase calcium mobilization via phospholipase C [32, 33]. In rat AR42J cells, SSTR2 mediated phospholipase C-independent Ca2/ mobilization by opening cell-surface calcium channels [34]. Somatostatin and its analogues has been used to inhibit growth of tumor cells derived from pancreas [35], breast [36, 37], colon [38], thyroid [39], prostate [40], lung [41], and pituitary and brain [42]. The inhibition of tumor cell growth by somatostatin was related to the activity of phosphotyrosine phosphatase [36, 43]. Recently, it have been reported that in trans-
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fected hSSTR2 cells such as COS-7 and NIH 3T3, somatostatin analogues RC-160 and SMS 201-995 inhibited transfected cell growth by stimulating tyrosine phosphatase activity [44]. The pharmacological characteristics of somatostatin receptor type 2 for various of somatostatin analogues have been studied in different species using labeled somatostatin and its analogues. SSTR2 cloned from mouse, rat, and human tissues exhibited high and similar affinity for somatostatin-14 and somatostatin-28 in transfected CHO-G44, CHO-K1, and COS1 cells. [14, 23, 25]. The 125I-MK-678 (a somatostatin hexapeptide analogue) binding to mouse SSTR2 was inhibited by somatostatin and its analogue RC 160 in the transfected CHO cells [45, 46]. hSSTR2 bound with labeled somatostatin-28 was inhibited by somatostatin analogue RC-160 and SMS 201-995 (Octreotide) in transfected CHO-K1 cells [25]. RC-160 and SMS 201-995 were also used in the studies of the other SSTR subtypes. RC-160 had high binding affinity for SSTR3-5, but not SSTR1 [25]. SMS 201-995 had high binding affinity for SSTR3 and SSTR5, but not for SSTR1 and SSTR4 [25, 46]. In this study, the receptor binding affinity of transfected HEK and CHO cells was studied using somatostatin-14, somatostatin-28, RC-160, and NC-9-74 which is specific for SSTR2, and DC-37-39 which is specific for SSTR5 [25, 47]. The two stable transfected cell lines showed high affinity for somatostatin-14, somatostatin-28, somatostatin analogue RC-160, and SSTR2specific agonist NC-9-74. SSTR2 had binding affinity for NC-9-74 similar to that of somatostatin-14 and somatostatin-28, confirming that NC-9-74 was a specific agonist for SSTR2. The close IC50 values of two stable cell lines binding the somatostatin analogues reflected that there is no difference in binding affinity of the two types of transfected cells. On the other hand, DC-3739, a selective SSTR5 agonist, bound both HEK and CHO with very low affinity, indicating that there is a pharmacological difference between SSTR2 and SSTR5. These data demonstrated that the transfected CHO and HEK cells have a high affinity for the SSTR2 agonist but not for the SSTR5 agonist. In the cell growth study, we found for first time that SSTR2-specific agonist NC-9-74 inhibited growth of HEK cells stably transfected with human SSTR2 in the medium containing 10% fetal bovine serum, but not stably transfected CHO cells. These data imply that hSSTR2 has an antiproliferative role and that HEK cells have the intracellular environment for allowing SSTR2 to mediate cell antiproliferation, whereas the CHO cells do not. The latter could be explained by the lack of the proper G-protein (Gia1) in the CHO cells, since RC 160 was able to inhibit CHO cell proliferation when these cells were transfected with mouse SSTR2 with G-protein Gia1 [48]. In contrast, somatostatin failed to increase tyrosine phosphatase activity in CHO-K1 transfected with rat SSTR2 [49]. In conclusion, the results of the present study demonstrate that human somatostatin receptor type 2 can be
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transfected into HEK and CHO cells and that these stable cell lines show binding similar to somatostatin analogues. SSTR2-specific agonist NC-9-74 significantly inhibited growth of transfected HEK cells but not CHO cells, suggesting that activation of SSTR2 was more efficiently coupled to the signal transduction pathway of antiproliferation in the transfected HEK cells than in the CHO cells. This study confirms the specificity of the SSTR2 agonist for the human SSTR2 receptor. Further studies are necessary to determine the signal transduction pathway of antiproliferation in the transfected SSTR cells. ACKNOWLEDGMENT This work was supported by NIH Grant R29DK46441-01.
REFERENCES 1. Reichlin, S. Somatostatin. N. Engl. J. Med.309: 1495, 1983. 2. Reisine, T., and Bell, G. I. Molecular properties of somatostatin receptors. Neuroscience 67: 777, 1995. 3. Patel, Y. C., Greenwood, M. T., Panetta, R., Demchyshyn, L., Niznik, H., and Srikant, C. B. Mini review the somatostatin receptor. Fam. Life Sci. 57: 1249, 1995. 4. Bloom, S., Mortimer, C., Thorner, M., Besser, G., Hall, R., et al. Inhibition of gastrin and gastric acid secretion by growth hormone release-inhibitory hormone. Lancet 2: 1106, 1974. 5. Tannenbaum, G., Ling, N., and Brazeau, P. SRIF 28 is longer acting and more selective than SRIF on pituitary and pancreatic hormone release. Endocrinology 111: 101, 1982. 6. Gomez-pan, A., Reed, J., Albinus, M., Shaw, B., et al. Direct of gastric acid and pepsin secretion by growth-hormone releaseinhibiting hormone in rat. Lancet 1: 888, 1975. 7. Epelbaum, J. Somatostatin in the central nervous system: Physiology and pathological modification. Prog. Neurobiol. 27: 63, 1986. 8. Brazeau, P., Vale, W., Burgus, R., Ling, N., Rivier, J., and Guillemin, R. Hypothalamic polypeptide that inhibits the secretion of immunoreactive pituitary growth hormone. Science 129: 77, 1973. 9. Pradayrol, L., Jornvall, H., Mutt, V., et al. N-terminally extended somatostatin: The primary structure of somatostatin28. FEBS Lett. 109: 55, 1980. 10. Schally, A. V. Oncological application of somatostatin analogues. Cancer Res. 48: 6977, 1988. 11. Vanetti, M., Kouba, M., Wang, X., Vogt, G., and Hoellt, V. Cloning and expression of a novel mouse somatostatin receptor (SSTR2B). FEBS Lett. 311: 290, 1992. 12. Patel, Y. C., Greenwood, M., Kent, B., Panetta, R., and Srikant, C. B. Multiple gene transcripts of the somatostatin receptor SSTR2: Tissue selective distribution and cAMP regulation. Biochem. Biophys. Res. Commun. 192: 288, 1993. 13. Taylor, J. E., Theveniau, M. A., Bashirzadeh, R., Resine, T., and Eden, P. A. Detection of somatostatin receptor subtype 2 (SSTR2) in established tumors and tumor cell lines: Evidence for SSTR2 heterogeneity. Peptides 15: 1229, 1994. 14. Yamada, Y., Post, S. R., Wang, K., et al. Cloning and functional characterization of a family of human and mouse somatostatin receptors expressed in brain, gastrointestinal tract, and kidney. Proc. Natl. Acad. Sci. USA 89: 251, 1992. 15. Yamada, Y., Reisine, T., Law, S. F., et al. Somatostatin receptors, an expanding Gene family: Cloning and human SSTR3, a protein coupled to adenylyl cyclase. Mol. Endocrinol. 6: 2136, 1992.
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16. Yamada, Y., Kagimoto, S., Kubota, A., et al. Cloning, functional expression and pharmacological characterization of a fourth (hSSTR4) and a fifth (hSSTR5) human somatostatin receptor subtype. Biochem. Biophys. Res. Commun. 195: 844, 1993. 17. Rohrer, L., Raulf, F., Bruns, C., et al. Cloning and characterization of a fourth human somatostatin receptor. Proc. Natl. Acad. Sci. 90: 4196, 1993. 18. Panetta, R., Greenwood, M. T., Warszynska, A., et al. Molecular cloning, functional characterization and chromosomal localization of a human somatostatin receptor (somatostatin receptor type 5) with preferential affinity with somatostatin-28. Mol. Pharmacol. 45: 417, 1994. 19. Breder, C. D., Yamada, Y., Yasuda, K., Seino, S., Saper, C., and Bell, G. I. Differential expression of SRIF receptor subtypes in brain. J. Neurosci. 12: 3920, 1992. 20. Bruno, J. F., Xu, Y., Song, J., and Berelowitz, M. Tissue distribution of SRIF receptor subtype messenger ribonucleic acid in the rat. Endocrinology 133: 2561, 1993. 21. Kong, H., DePaoli, A. M., Breder, C. D., Yasuda, K., Bell, G. I., and Reisine, T. Differential expression of SRIF receptor subtypes SSTR1, SSTR2 and SSTR3 in adult rat brain, pituitary and adrenal gland. Analysis by RNA blotting and in situ hybridization. Neuroscience 59: 175, 1994. 22. Raulf, F., Perez, J., Hoyer, D., and Bruns, C. Differential expression of five somatostatin receptor subtypes, SSTR1-5, in the CNS and peripheral tissue. Digestion 55: 46, 1994. 23. Kluxen, F. W., Bruns, C., and Luebbert, H. Expression and cloning of a rat brain somatostatin receptor cDNA. Proc. Natl. Acad. Sci. 89: 4618, 1992. 24. O’Carroll, A. M., Lolait, S. J., Konig, M., and McMahan, L. C. Molecular cloning and expression of a pituitary somatostatin receptor with preferential affinity for somatostatin-28. Mol. Pharmacol. 42: 939, 1992. 25. Patel, Y. C., and Srikant, C. B. Subtype selectivity of peptide analogs for all five cloned human somatostatin receptors (hSSTR1-5). Endocinology 135(6): 2814, 1994. 26. Moldovan, S., Atiya, A., Adrian, T. E., Kleinman, R. M., et al. Somatostatin inhibits B-cell secretion via a subtype-2 somatostatin receptor in the isolated perfused human pancreas. J. Surg. Res. 59: 85, 1995. 27. Kaupmann, K., Bruns, C., Hoyer, D., Seuwen, K., and Luebbert, H. Distribution and second messenger coupling of four somatostatin receptor subtypes expressed in brain. FEBS Lett. 331: 53, 1993. 28. Strnad, J., Eppler, C. M., Corbett, M., and Hadcork, J. R. The rat SSTR2 somatostatin rceptor subtype is coupled to inhibition of cyclic AMP accumulation. Biochem. Biophys. Res. Commun. 191: 968, 1993. 29. Yasuda, K., Rens-Domiano, S., Breder, C.D., et al. Cloning of a novel somatostatin receptor, sstr3, coupled to adenylyl cyclase. J. Biol. Chem. 267: 20422, 1992. 30. Raynor, K., O’Carroll, A. M., Kong, H. Y., et al. Characterization of cloned somatostatin receptors SSTR4 and SSTR5. Mol. Pharmacol. 44: 385, 1993. 31. Patel, Y. C., Greenwood, M. T., Warszynska, A., Panetta, R., and Srikant, C. B. All five cloned human somatostatin rceptors (hSSTR1-5) are functionally coupled to adenylyl cyclase. Biochem. Biophys. Res. Commun. 198: 605, 1994. 32. Tomura, H., Okajima, F., Akbar, M., Majid, M. A., Sho, K., and Kondo, Y. Transfected human somatostatin receptors type 2, SSTR2, not only inhibits adenylate cyclase but also stimulates phospholipase C and Ca2/ mobilization. Biochem. Biophys. Res. Commun. 200: 986, 1994. 33. Akbar, M., Okajima, F., Tomura, H., Majid, M. A., Yamada, Y., Seino, S., and Kondo, Y. Phospholipase C activation and Ca2/ mobilization by cloned human somatostatin receptor subtypes 1-5, in transfected COS-7 cells. FEBS Lett. 348: 192, 1994. 34. Taylor, J. E. Somatostatin (SSTR2) receptors mediate phospho-
srga
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35.
36.
37.
38.
39.
40.
41.
42.
AID
JOURNAL OF SURGICAL RESEARCH: VOL. 71, NO. 1, JULY 15, 1997 lipase C-independent Ca2/ mobilization in rat AR42J pancreas cells. Biochem. Biophys. Res. Commun. 214: 81, 1995. Liebow, C., Reilly, C., Serrano, M., and Schally, A. V. Somatostatin analogues inhibit growth of pancreatic cancer by stimulating tyrosine phosphatase. Proc. Natl. Acad. Sci. USA 86: 2003, 1989. Setyono-Han, B., Henkelman, M. S., Foekens, J. A., and Klijn J. G. M. Direct inhibitory effects of somatostatin (analogues) on the growth of human breast cancer cell lines. Cancer Res. 47: 1566, 1987. Scambia, G., Panici, P. B., Baiocchi, G., Perrone, L., Oacobelli, S., and Mancuso, S. Antiproliferative effects of somatostatin and the somatostatin analog SMS 201-995 on three human breast cancer cell lines. J. Cancer Res. Clin. Oncol. 114: 306, 1988. Qin, Y., Schally, A. V., and Willems, G. Somatostatin analogues RC-160 inhibits the growth of transplanted colon cancer in rats. Int. J. Cancer 47: 765, 1991. Tsuzaki, S., and Moses, A. C. Somatostatin inhibits deoxyribonucleic acid synthesis induced by both thyrotropin and insulinlike growth factor-1 in FRTL5 cells. Endocrinology 126: 3131, 1990. Schally, A. V., and Redding, R. W. Somatostatin analogs as adjuncts to agonist of luteinizing hormone-releasing hormone in the treatment of experimental prostate cancer. Proc. Natl. Acad. Sci. USA 84: 7275, 1987. Taylor, J. E., Moreau J. P., Baptiste, L., and Moody, T. W. Octapeptide analogues of somatostatin inhibit the clonal growth and vasoactive intestinal peptide-stimulated cyclic AMP formation in human small cell lung cancer cells. Peptides 12: 839, 1990. Lamberts, S. W. J., Krenning, E. P., and Reubi, J. C. The role of somatostatin and its analogs in the diagnosis and treatment of tumors. Endocrinol. Rev. 12: 450, 1991.
JSR 5097
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6n21$$$321
07-18-97 15:34:12
43. Lee, M. T., Liebow, C., Kamer, A. R., and Schally, A. V. Effects of epidermal growth factor and analogues of luteinizing hormone-releasing hormone and somatostatin on phosphorylation and dephosphorylation of tyrosine residues of specific protein substrates in various tumors. Proc. Natl. Acad. Sci. USA 88: 1656, 1991. 44. Buscail, L., Delesque, N., Esteve, J. P., Saint-Laurent, N., et al. Stimulation of tyosine phosphatase and inhibition of cell proliferation by somatostatin analogues: Mediation by human somatostatin receptor subtypes SSTR1 and SSTR2. Proc. Natl. Acad. Sci. USA 91: 2315, 1994. 45. Rens-Domiano, S., Law, S. F., Yamada, Y., Seino, S., Bel, G. I., and Reisine, T. Pharmacological properties of two cloned SRIF receptors. Mol. Pharmacol. 42: 28, 1992. 46. Raynor, K., Murphy, W. A., Coy, D. H., et al. Cloned somatostatin receptors: Identification of subtype-selective peptides and demonstration of high affinity binding of linear peptides. Mol. Pharmacol. 43: 838, 1993. 47. Rossowski, W. J., and Coy, D. H. Specific inhibition of rat pancreatic insulin or glucagon release by receptor-selective somatostatin analogs. Biochem. Biophys. Res. Commun. 205(1): 341, 1994. 48. Buscail, L., Esteve, J. P., Saint-Laurent, N., Bertrand, V., Reisine, T., O’Carroll, A. M., Bell, G. I., Schally, A., Vaysse, N., and Susini, C. Inhibition of cell proliferation by the somatostatin analogue RC-160 is mediated by SSTR2 and SSTR5 somatostatin receptor subtypes through different mechanisms. Proc. Natl. Acad. Sci. USA 92: 1580, 1995. 49. Florio, T., Rim, C., Hershberger, R. E., Loda, M., and Stork, P. J. The somatostatin receptor SSTR1 is coupled to phosphotyrosine phosphatase activity in CHO-K1 cells. Mol. Endocrinol. 8(10): 1289, 1994.
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