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Original Paper

Cell Physiol Biochem 2002;12:31-38 Accepted: November 30, 2001Cellular PhysiologyCellular PhysiologyCellular PhysiologyCellular PhysiologyCellular Physiologyand Biochemistrand Biochemistrand Biochemistrand Biochemistrand Biochemistryyyyy

Copyright © 2002 S. Karger AG, Basel

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Activation of Human Somatostatin Receptor 2Promotes Apoptosis Through a Mechanism thatis Independent from Induction of p53

Rosa Teijeiro, Ramón Ríos, José A. Costoya, Ramón Castro, JoséL. Bello, Jesús Devesa and Víctor M. Arce

Departamento de Fisioloxía. Facultade de Medicina. Universidade de Santiago de Compostela

Víctor M. ArceDepartamento de Fisioloxía, Facultade de MedicinaRua San Francisco s/n, 15705 Santiago de Compostela (Spain)Tel. +34981582658, Fax +34981574145E-Mail fsvarce@usc.es

Key WordsSomatostatin • Somatostatin receptor • Apoptosis •p53 • p63 • p73

AbstractThe ability of both somatostatin (SS) and its stableanalogues to inhibit cell growth depends on the stimu-lation of specific membrane receptors (SSTR1-5),which belong to the G protein-coupled receptor fam-ily. Accumulating evidence suggests that the SSTR2plays a major role in mediating cell cycle arrest, and itis also clear that SHP-1, a cytoplasmicphosphotyrosine phosphatase (PTP), is an essentialcomponent of the SSTR2-mediated cytostatic effect.In contrast, the possibility that SSTR2 activation mayalso lead to increased apoptosis is still beyond de-bate, despite SHP-1 activation is also able to promotecell death in several cell types. In the present workwe have investigated the ability of SSTR2 to induceapoptosis in HL-60 cells. We have found that HL-60cells uniquely express the SSTR2 subtype, and thatstimulation of SSTR2 with the SS analogue SMS 201-995 results in an increased cell death. In all, these

findings demonstrate that activation of SSTR2 pro-motes apoptosis in HL-60 cells. Moreover, in contrastwith the proapoptotic mechanism previously reportedfor SSTR3, cell death induced by activation of SSTR2is independent from accumulation of p53.

Introduction

Somatostatin (SS) is a widely distributed polypep-tide, first isolated from the hypothalamus as an inhibitorof GH secretion, but later demonstrated in other regionsof the central nervous system as well as in peripheraltissues such as the kidneys, the pancreas, thegastrointestinal tract and the hemopoietic and immunesystems [1-3]. The physiological effects of SS are pre-dominantly inhibitory. SS can reduce both exocrine andendocrine secretions and inhibits cell proliferation. Inaddition, SS analogues can also stimulate apoptosis inseveral cellular types [4, 5]. All these actions are medi-

32 Teijeiro/Rios/Costoya/Castro/Bello/Devesa/Arce

ated by interacting with five different types of SSreceptors, named SSTR1 through SSTR5, which belongto the G protein coupled receptor family. Binding of SSor SS analogues to their receptors activate a number ofG protein-linked signal transduction pathways, includ-ing adenylyl cyclase (SSTR1-5), phosphotyrosine phos-phatase (PTP) (SSTR1, -2, and -3), Ca2+ channels(SSTR2), phospholipase A2 (SSTR4), and mitogen-acti-vated protein kinase (SSTR4) [3, 6].

Although the involvement of each receptor subtypein SS responses remains to be clarified, accumulatingevidence suggests that SSTR2 plays a major role in me-diating cell cycle arrest in various cellular types [7-10].This effect appears to be largely mediated by SHP-1 [10],a cytoplasmic PTP that contains two Src homology 2(SH2) domains involved in its association with multiplesignaling systems including tyrosine kinase receptors,cytokine receptors and G-protein coupled receptors [11].SSTR2-induced activation of SHP-1 results in inductionof the cyclin-dependent kinase (cdk) inhibitor p27kip1 andconsequently, an inhibition of cyclinE-cdk2 kinase ac-tivity and cell arrest [10].

In contrast, the possibility that stimulation of SSTR2leads to cell death is still under debate. In CHO cellsstably expressing each of the individual SSTRs, apoptosiswas uniquely signaled through SSTR3 [12]. However, itis also well established that, besides its role in inducingcell arrest, activation of SHP-1 can promote cell death[13]. Moreover, this cytotoxic effect can be triggered bySSTR stimulation, although the SSTR subtype involvedhas not been elucidated so far [13]. Therefore, sincestimulation of SHP-1 is an essential component of SSTR2signaling, in the present work we investigated the possi-bility that SSTR2 activation could also induce a cyto-toxic effect.

Materials and Methods

Cell culture and treatmentsExperiments were carried out in HL-60 cells, provided by

the European Collection of Animal Cell Cultures. Cells weregrown in Falcon polystyrene culture flasks (Becton DickinsonS.A., Madrid, Spain), containing RPMI-1640 medium (InvitrogenS.A., Barcelona, Spain) supplemented with 10% fetal calf serum(Invitrogen) and 2 mM L-glutamine (Sigma Química, Madrid,Spain), at 37ºC in 5% humidified CO

2.

Induction of apoptosis was achieved by growing the cellsfor 4 days in serum-free media, in the presence of different doses(9·10-8 M, 9·10-9 M, 9·10-10 M, 9·10-11 M, 9·10-12 M, or 9·10-13M) of

the SS analogue SMS 201-995 (Novartis, Basel, Switzerland). Ineach case, cell number was determined with a Neubauer cham-ber.

RT-PCRPoly A+ RNA was isolated with a commercial kit (Direct

mRNA Isolation Kit; CPG Inc, Lincoln Park, NJ), and reverse-transcribed for 1 hour at 37 oC with 200 U of M-MLV reversetranscriptase (Invitrogen), followed by 5 min at 95 oC, in a 30 µl-reaction mixture containing 50 mM Tris-HCl (pH 8.3), 75 mMKCl, 5.5 mM MgCl

2, 0.5 mM each dNTP, 40 U of RNaseOUT

recombinant ribonuclease inhibitor (Invitrogen) and 1.7 µg/µl ofrandom primers (Invitrogen). Amplification of cDNA was per-formed using gene-specific 20 bp oligonucleotide primers (25pM each) (see table 1), 0.2 mM dNTPs, 1.25 units of Taq-DNAPolymerase (Invitrogen) and 5 µl of 10X PCR buffer (18.6 mMTris-HCl, 45.9 mM KCl, 3 mM MgCl

2), in a total volume of 50µl.

Reactions were overlaid with mineral oil and put through 30 cy-cles of denaturation for 1 min at 94ºC, annealing for 1 min, andelongation for 1 min at 72ºC; followed by final elongation cyclefor 10 min at 72ºC. In order to better assess the specificity of theamplimers, several annealing temperatures (53, 58, 63, and 68ºC)were used for each pair of primers. Negative control was per-formed by omitting the reverse-transcription. Human genomicDNA (isolated from peripheral blood cells) was used as positivecontrol for each SSTR subtype. In this case, 24 cycles of amplifi-cation were performed, and the annealing temperature was set at60ºC. Identity of amplimers was confirmed by restriction enzymecleavage with MspI (APBiotech, Barcelona, Spain), which spe-cifically recognizes a sequence within the SSTR2 gene. Allamplimers were resolved in 1% agarose gels, stained with ethidiumbromide and visualized with a Gel Doc system (Bio-Rad Labora-tories Inc., Hercules, CA).

Assessment of apoptosisApoptotic cell death was assessed by microscopic analysis

of cell DNA staining patterns with Hoechst 33258 (SigmaQuímica). Cells were incubated with 20 µg/ml of Hoechst 33258for 10 min at room temperature and, after washing, cell morphol-ogy was examined under an Olympus fluorescence microscope(IX70, Olympus Optical Co, Tokyo, Japan) with the appropriatefilter combination. Cells were scored for apoptosis by their nuclearmorphology (shrinkage, condensation, and fragmentation) and thehigher intensity of blue fluorescence and photographed at a 40Xmagnification with a DP10 microscope digital camera (OlympusOptical Co).

Western blottingCells were collected by centrifugation (100 xg, 3 min, 4ºC),

resuspended in ice-cold PBS, and centrifuged again. The result-ing pellet was resuspended in 200 µl lysis buffer (10 mM Tris.·HClpH 7.6, 5 mM EDTA, 150 mM NaCl, 10% glycerol, 0.5% TritonX-100, 30 mM sodium pyrophophate, 50 mM NaF, 1 mM sodiumorthovanadate, aprotinin and leupeptin) and incubated for 15minutes on ice. The cell extracts were then centrifuged to pelletthe cell debris and the supernatants frozen at -80C. Bradford re-agent (Bio-Rad Laboratories) was used to measure total protein

Cell Physiol Biochem 2002;12:31-38

33Activation of Human Somatostatin Receptor 2 Promotes Apoptosis

Fig. 1. HL-60 cells express only the SSTR2 subtype. A) RT-PCR assay for SSTR subtypes 1-5 (221, 213, 235, 235 and191 bp) of mRNA isolated from HL-60 cells. As the SSTRslack introns, amplification of human genomic DNA (GN)served as a positive control for each of the SSTR subtypes.During the amplification of cDNA, several hybridization tem-peratures (hyT) where tested for each of the primer pairs. Onlyprimers for SSTR2 isoform yielded a specific PCR productwhen increasing hyT. In contrast, the magnitude of the otherSSTR products decreased as the hyT increased, thus suggest-ing an unspecific amplification. B) (Left panel) Negative con-trol, showing that no PCR product is present if the mRNAsamples are not reversed transcribed. (Right panel) Restric-tion analysis of the PCR product for SSTR2 with MspI.

Table 1: Sequences of thedifferent sets of primers de-signed to specifically amplifyeach SSTR subtype. The pre-dicted size of each product isalso indicated.

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content. 50 µg of total protein was separated by SDS-PAGE (7.5%)and electrotransferred onto a nitrocellulose paper (Protran;Schleicher and Schuell, Dassel, Germany). The membranes wereblocked at 4oC overnight in Tris-buffered saline buffer (TBS) with0.1% Tween20 and 0.25% gelatin and then incubated for 1 hourat room temperature with a purified mouse anti-human p73 anti-body (Becton Dickinson S.A) which recognizes the α and βisoforms of human p73. The membranes were then washed withTBS-Tween 0.1% and further incubated with Protein A-horse-radish peroxidase (HRP) conjugate (APBiotech) at 1 :1500 dilu-tion for 1 hour at room temperature. After washing, HRP activitywas detected with a western-light chemiluminiscence detectionsystem (ECL, APBiotech) and photographed (HyperfilmECL,APBiotech).

Statistical analysisStatistical analysis was performed with the non-parametric

Mann-Whitney test. Statistical significance was established atp<0.05.

Fig. 2. Effect of SMS 201-995 treatment on HL-60 cell growth.Cells were serun-starved, treated with 9·10-10M of SMS 201-995for 24, 48 or 72 hours, and counted in a Neubauer chamber. Eachbar represents the mean+SEM of 4 independent experiments intriplicate. **=p<0.01 vs control.

Fig. 3. Effect of SMS 201-995treatment on HL-60 cell survival.Cells were serun-starved andtreated with 9·10-10M of SMS201-995 for 24, 48 or 72 hours.A) Fluorescent microscopicanalysis of cell DNA staining pat-terns with Hoechst 33258. Cellswere scored for apoptosis by theirnuclear morphology (shrinkage,condensation, and fragmentation)and the higher intensity of bluefluorescence. Each bar representsthe mean+SEM of 4 independentexperiments in triplicate.**=p<0.01 vs control. B) Repre-sentative photograph of the resultspresented in panel A. Lensobjetive 40X.

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Fig. 4. SMS 201-995 treatment does not induce p73 accumula-tion in HL-60 cells. Cells were serun-starved and treated with9·10-10M of SMS 201-995 for 24, 48 or 72 hours. Cells extractswere resolved in a 7.5% SDS-PAGE, and electrotransferred ontoa nitrocellulose paper. Membranes were then probed with a mouseanti-human p73 antibody which reacts with the α and β isoformsof p73. The p73β isoform, which migrates at molecular weightsof approximately 70 kD, is shown (arrow).

Results

HL-60 cells express uniquely the SSTR2 subtypeIn order to assess which SSTR subtype is being ex-

pressed by HL-60 cells, RT-PCR amplifications wereperformed with oligonucleotide primers designed to spe-cifically amplify each receptor subtype. Amplificationof cDNA from HL-60 cells only yielded a specific prod-uct with the primers corresponding to the SSTR2 se-quence. In this case, a band migrating at the same sizethat the genomic control, and in keeping with the pre-dicted size (213 bp) of the amplified fragment could beobserved (Fig. 1A). In contrast, the bands obtained withthe other sets of primers showed migrating sizes differ-ent to those expected. Moreover, an increase in band in-tensity when augmenting the hybridization temperaturewas only observed with SSTR2 primers, with the maxi-mal amplification obtained with the highest annealingtemperature tested (68ºC). In contrast, the magnitude ofthe other products decreased as the hybridization tem-perature increased, thus suggesting an unspecific ampli-fication (Fig 1A).

The possibility that the SSTR2 band could be origi-nated by amplification of any genomic DNA present inthe sample was ruled out according with the results ob-tained when no reverse transcription was performed (Fig1B). Finally, enzymatic digestion of the RT-PCR prod-uct with Msp-I originated two bands migrating at about154 and 59 bp, also in keeping with the predicted results(Fig. 1B). This finding further confirms the identity ofthe product, since the sequence recognized by Msp-I ispresent in the amplified fragment of the SSTR2 gene,but not in the other members of the SSTR gene family.

SMS 201-995 treatment induces apoptosis of HL-60 cellsAmong the different SMS 201-995 doses tested, the

9·10-10M was de most effective in inducing the apoptoticdeath of HL-60 cells (data not shown). As shown in fig-ure 2, treatment of HL-60 cells with this dose of SMS201-995 induced a significant reduction of cell growth.Cell counts on day 3 were 26.580±3.200 in control cellsvs. 20.170±3.400 in SMS 201-995-treated cells (p<0.05).Fluorescent microscopic analysis of cell DNA stainingpatterns with Hoechst 33258 revealed also a significantincrease in apoptotic cell death in SMS 201-995-treatedcells as compared with control cells (figure 3).

p73 levels do not increase during SMS 201-995-induced apoptosis

Accumulation of p73 in HL-60 cells was determinedby western blotting (figure 4). Serum starvation inducedan increase in p73 immunoreactivity, that achieved itsmaximal intensity after 72 h. In contrast, no differenceswere observed between SMS 201-995- or vehicle-treatedcells at any time investigated.

Discussion

Together with its ability to inhibit multiple endo-crine and exocrine secretions, SS exerts direct anti-pro-liferative effects with cytostatic (growth arrest) or cyto-toxic (apoptosis) consequences. The cytostatic effect ofboth SS and its stable analogs has been demonstrated inmultiple cell types, including GH3 (rat pituitary tumorcells), AtT20 (mouse pituitary tumor cells), MCF-7 (hu-man breast adenocarcinoma cells), PANC-1 (human pan-creatic cancer), PC-1.0 (hamster pancreatic ductal carci-noma), FRTL-5 (rat thyroid carcinoma), Jurkat (humanT-cell leukemia) and in several human multiple myelomacell lines [7, 14-19].

The mechanism by which SS inhibits cell growthhas not been completely elucidated to date, mainly due

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to the existence of five distinct SSTRs which regulatemultiple signaling pathways, and to the fact that mostcells express more than one SSTR subtype. There is,however, strong evidence indicating that SSTR2 may playa major role in SS-induced cell arrest. Studies performedin CHO cells stably expressing the cDNA encoding thehuman SSTR2 sequence demonstrated that treatmentwith SS analogues such as SMS 201-995 or RC-160 in-hibits serum- or insulin-induced cell growth [8, 10, 12].The stable expression of SSTR2 also inhibits the prolif-eration of PC-1 cells in vitro, as well as the metastaticprogression of these cells after intrapancreatic implan-tation [7]. Finally, the loss of SSTR2 expression has beenreported to be a growth advantage in human pancreaticand colorectal cancer [9].

Some of the cell cycle regulatory events leading toSSTR-mediated growth arrest have been recently eluci-dated. Using CHO cells expressing SSTR2, it has beenshown that SHP-1 is an essential component of SSTR2signaling [20]. Upon SSTR2 stimulation, SHP-1 disso-ciates from SSTR2, resulting in an increase in SHP-1activity. Once activated, SHP-1 inhibits cell prolifera-tion by terminating mitogenic signals, which results ininduction of p27kip1, a member of the p21 family cyclin-dependent kinase (cdk) inhibitors [21]. This rise in p27kip1

increases p27kip1-cdk2 association and, subsequently, re-duces cyclin E-cdk2 activity. The final consequence ofthese changes is the accumulation of hypophosphorylatedretinoblastoma protein (pRb) and growth arrest [10].

In addition to inducing growth arrest, SHP-1 is alsorequired for regulating multiple apoptotic events, includ-ing somatostatin-signaled cell death in MCF-7 humanbreast cancer cells [13]. Therefore, since SHP-1 is anessential component of SSTR2 signaling, it is temptingto speculate that activation of SSTR2 may also induce acytotoxic effect. In keeping with this hypothesis, wefound, in the present work, that activation of SSTR2 bymeans of SMS 201-999 treatment induced apoptosis inHL-60 cells, a cell line that uniquely express the SSTR2subtype.

Although the mechanism underlying the cytotoxiceffect of SHP-1 is not completely understood, it involvesthe accumulation of p53, a tumor-suppressor protein thattriggers G1 arrest, but also induces apoptosis when a de-fective or inappropriate cell cycle progression occurs[13]. The increase in p53 induces the accumulation ofBax, a pro-apoptotic member of the Bcl-2 family of pro-

teins that is in part responsible for p53-induced apoptosis[22]. Interestingly, both SS and SMS 201-995 inducewild-type p53 and Bax in MCF-7 cells (although theSSTR subtype involved in this case could not be identi-fied because MCF- 7 cells express multiple SSTR sub-types) and in CHO cells stably expressing the SSTR3cDNA [4, 12].

However, the accumulation of p53 cannot explainthe ability of SMS 201-995 to promote apoptosis in HL-60 cells, since these cells have a null p53 phenotype be-cause of a large deletion of the p53 gene [23]. Severalmechanisms may explain how SSTR2 activation mayinduce apoptosis in absence of p53. The first possibilityis that stimulation of SSTR2 induces the accumulationof another member of the p53 family of transcriptionfactors which also includes p63 and p73 [24]. Despitethe homology shared, p53 family of transcription fac-tors exert distinct biological functions ranging from tu-mor suppression to regulation of development. In thispicture, the primary biological function of p63 protein isto regulate differentiation, and does not play a role simi-lar to p53 in the control of cancer [26]. In contrast, p73can activate p53-regulated genes and induce apoptosisin some instances, even in the absence of p53 [27, 28].Therefore, it appears that p73 is the candidate to pro-mote apoptosis in cells lacking p53. Unfortunately, wehave been unable to find any change in p73 levels inHL-60 cells after SMS 201-995 treatment, thus suggest-ing that, at least under our experimental conditions, p73does not appear to contribute significantly to apoptosis.

Another possible mechanism underlying the cyto-toxic effect of SMS 201-995, depends on the ability ofSHP-1 to directly terminate survival pathways. Strongevidence indicates that SHP-1 is key regulator in my-eloid cell survival as revealed in motheaten mice (inwhich SHP-1 activity is essentially absent), or in cellsstably expressing enzimatically inactive forms of SHP-1[29, 30]. Although putative pro-apoptotic molecules thatare activated by SHP-1 through dephosphorylation havenot been identified to date, SHP-1 is known to associateto (and inactivate) receptor molecules that stimulate cellsurvival mechanisms, including insulin receptor and sev-eral members of the cytokine receptor superfamily suchas interleukine-3 receptor, erythropoietin receptor, orinterferon-α/β receptor [29, 31, 32] that play a majorrole in promoting the survival of hemopoietic cells. Dis-ruption of survival signals proceeding from these recep-

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tors may result in activation of Bad, another proapoptoticmember of the Bcl-2 family, and cytotoxicity [22, 29].

In summary our findings demonstrate that activa-tion of SSTR2 leads to apoptosis of HL-60 cells in vitro.While further studies are needed to ascertain the signal-ing pathways underlying this effect, it is clear that it is

not dependent on accumulation of either p53 (as occurswith the cytotoxic effect of SSTR3) or p73. Therefore,at least two different proapoptotic pathways can be acti-vated by SS or SS analogs: a p53-dependent pathwaythat is activated by SSTR3, and a p53-independent path-way triggered by SSTR2.

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