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Endocrinology, doi:10.1210/en.2005-1479
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Endocrinology Vol. 147, No. 7 3530-3538
Copyright © 2006 by The Endocrine Society

Role of Complex Cyclin D1/Cdk4 in Somatostatin Subtype 2 Receptor-Mediated Inhibition of Cell Proliferation of a Medullary Thyroid Carcinoma Cell Line in Vitro

Federico Tagliati, Maria Chiara Zatelli, Arianna Bottoni, Daniela Piccin, Andrea Luchin, Michael D. Culler and Ettore C. degli Uberti

Section of Endocrinology (F.T., M.C.Z., A.B., D.P., A.L., E.C.d.U.), Department of Biomedical Sciences and Advanced Therapies, University of Ferrara, I-44100 Ferrara, Italy; and Biomeasure Inc./IPSEN (M.D.C.), Milford, Massachusetts 01757

Address all correspondence and requests for reprints to: Ettore C. degli Uberti, M.D., Section of Endocrinology, Department of Biomedical Sciences and Advanced Therapies, University of Ferrara, Via Savonarola 9, 44100 Ferrara, Italy. E-mail: ti8{at}unife.it.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Somatostatin (SRIH) inhibits cell proliferation by interacting with five distinct SRIH receptor subtypes (SSTRs) activating several pathways in many tissues. We previously demonstrated that SRIH, by activating Src homology-2-containing protein, inhibits cell proliferation of the human medullary thyroid carcinoma cell line, TT, which expresses all SSTRs. However, the effects of SRIH on cell cycle proteins have not been investigated so far. We therefore evaluated the effects of SRIH and a selective SSTR2 agonist on cell cycle protein expression, mainly focusing on cyclin D1 and its associated kinases. Our data show that SRIH and the selective SSTR2 agonist, BIM-23120, reduce cell proliferation and DNA synthesis as well as induce a delay of the cell cycle in G2/M phase. Moreover, treatment with both SRIH and BIM-23120 decreases cyclin D1 levels, with a parallel increase in phosphocyclin D1 levels, suggesting protein degradation. Moreover, our data show an increase in glycogen synthase kinase-3ß activity, which triggers phosphorylation-dependent cyclin D1 degradation. Indeed, we observed a reduction in cyclin D1 protein half-life under treatment with SRIH or the SSTR2 selective agonist. A reduction in cdk4 protein levels is also observed with a parallel reduction in Rb phosphorylation levels at Ser-780. Our data indicate that the subtype 2 receptor-mediated antiproliferative effect of SRIH on TT cell proliferation may be exerted through a decrease in cyclin D1 levels.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
SOMATOSTATIN (SRIH) HAS potent inhibitory effects on cell function in many tissues, in which five distinct SRIH receptor subtypes (SSTRs) are expressed with different patterns (1). It has been shown that cell proliferation and secretory activity are affected by SRIH and its analogs, both in vitro and in vivo, via specific SSTR, triggering different postreceptor actions, depending on the tissue (2). SRIH analogs are able to control symptoms of many neuroendocrine tumors including medullary thyroid carcinoma (MTC), providing clinical benefit by improving the quality of life in these patients (3). However, these effects are not always observed (4, 5), and there is scant evidence that octreotide, a SRIH analog currently used in clinical practice, reduces the tumor mass or improves the patient survival rate (5, 6). We previously demonstrated that SRIH and SRIH analogs interacting with SSTR2 inhibit cell proliferation in a human MTC cell line, TT, which expresses all five SSTRs (7, 8). Moreover, we have shown that the inhibitory effects of SRIH on cell proliferation are mediated, at least in part, by a cytoplasmic protein tyrosine phosphatase, Src homology-2-containing protein (SHP)-1, which, in turn, is activated upon SRIH binding to SSTR2 (9). We also demonstrated that SHP-1 activation down-regulates MAPK signaling in TT cells, resulting in a decreased cell proliferation. It has been shown that cyclin D1 expression is positively regulated by the MAPK pathway (10). We therefore investigated the effects of SRIH and a selective SSTR2 agonist on cell cycle protein expression, mainly focusing on cyclin D1 and its associated kinases.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
All reagents, if not otherwise specified, were purchased from Sigma-Aldrich (Milano, Italy).

SRIH and SSTR selective agonist
SRIH (Stilamin 250) was purchased from Serono Pharma (Roma, Italy). The SSTR2 selective analog used in this study, BIM-23120, was provided by Biomeasure Inc. (Milford, MA), and used as previously reported (7). To prevent peptide degradation, we added 30 µM Bacitracin, as previously reported (11). At the concentration used in the present study, Bacitracin did not interfere with the assays and the cell function.

Cell culture
The TT cell line was obtained from the American Type Culture Collection (Manassas, VA), and maintained in culture in F-12 Ham’s medium (F-12), as previously described (7). For synchronization experiments, TT cells were grown to 50% confluence, rinsed twice with PBS, and partially synchronized in G0/G1, starving the cells in serum-free medium for 48 h. Cells were then released from starvation by adding 10% fetal bovine serum (FBS) without or with test substances at 10–8 M.

Cell viability
The effect of SRIH and of SSTR2 selective agonist on TT cell viability in vitro was assessed by the CellTiter 96 Aqueous nonradioactive cell proliferation assay (Promega Italia, Milano Italy), after 12, 24, 36, 48, 54, and 60 h of treatment without or with each compound at 10–8 M, as previously described (8, 9). Medium with or without treatments was renewed every 24 h.

DNA synthesis
The effect of SRIH and of SSTR2 selective agonist on TT cell DNA synthesis was assessed as previously described (7), by determining the rate of [3H]thymidine ([3H] thy) incorporation, in cells serum starved and treated for 12, 24, 36, 48, 54, and 60 h with 10% FBS without or with each compound at 10–8 M. Medium with or without treatments was renewed every 24 h. Radiolabeled thymidine was added to the cell culture 12 h before harvesting for radioactivity counting.

Growth curve studies
The effect of SRIH and the SSTR2 selective agonist on TT cell number was evaluated as previously described (12). Briefly, TT cells (2 x 104 cells/well) were plated in 24-multiwell plates, serum starved for 2 d, and then treated with 10% FBS without or with each compound at 10–8 M. Medium with or without treatments was renewed every 24 h. After 48, 54, and 60 h, cells were trypsinized and counted using a hemocytometer. Counts were obtained in at least six replicates.

Cell cycle analysis
TT cell cycle was investigated after a 48-h treatment with test substances by flow cytometry after ethanol-fixation and propidium iodide (PI) staining, as previously described (13). Briefly, after washing in PBS, 5 x 105 cells were fixed in 1 ml cold 70% ethanol at 4 C for at least 1 h. The cells were then centrifuged, washed twice in PBS, resuspended in 0.5 ml PBS, and treated with 0.1 µg RNase (type I-A) for 30 min at 37 C. PI (20 µg/ml) was subsequently added to each sample, which, after gentle mixing, was incubated in the dark at room temperature for 30 min. The PI fluorescence of individual nuclei was measured using a FACScan (Becton Dickinson, San Jose, CA). The proportions of cells in the G0/G1, S, and G2/M phase of the cell cycle were automatically calculated using Lysis II analysis software (Becton Dickinson). Experiments were carried out in triplicate for each data point and repeated at least six times.

Western blot analysis
After treatment with the test substances at different time points, cells were resuspended in sample buffer [60 mM Tris-HCl (pH 6.8), 10% glycerol, 2% sodium dodecyl sulfate] and lysed by boiling at 100 C for 5 min. Total protein cell extracts were measured by using the BCA protein assay reagent kit (Pierce Biotechnology Inc., Rockford, IL). Equal protein amounts (~100 µg) were fractionated on 10% SDS-PAGE and transferred by electrophoresis to nitrocellulose membranes (Schleicher & Schuell Italia SRL, Milano Italy). The membranes were incubated with 1:1000 monoclonal cyclin D1 antibody (BD PharMingen, Franklin Lakes, NJ; source: mouse; catalog no. 554181), phosphocyclin D1 (Thr286) antibody (Cell Signaling Technology, Beverly, MA; source: rabbit; catalog no. 2921L), polyclonal phosphoglycogen synthase kinase (GSK)-3ß (Ser 9) antibody (Cell Signaling Technology; source: rabbit; catalog no. 9336L), polyclonal cyclin E antibody (Calbiochem, San Diego, CA; source: rabbit; catalog no. PC438-500), monoclonal CDK4 antibody (Sigma-Aldrich; source: mouse; catalog no. C8218), monoclonal CDK6 antibody (Sigma-Aldrich; source: mouse; catalog no. C8343), 1:500 monoclonal GSK-3ß antibody (Sigma-Aldrich; source: rabbit; catalog no. G7914), 1:400 monoclonal cyclin D2 antibody (Sigma-Aldrich; source: mouse; catalog no. C 7339), and 1:100 polyclonal actin antibody (Sigma-Aldrich; source: rabbit; catalog no. A2066). Horseradish peroxidase-conjugated antirabbit IgG (Dako, Milano, Italy) and antimouse IgG (Dako) secondary antibodies were used at 1:2000, and binding was revealed using enhanced chemiluminescence Western blotting detection reagents (Amersham Biosciences, Little Chalfont Buckingamshire, UK). For SSTR2 expression analysis, SDS-PAGE of total TT cell lysates were probed with a polyclonal SSTR2 antibody (Gramsch Laboratories, Schwabhausen, Germany; source: rabbit; catalog no. SS-860) used at 1:1000. Detection was performed as described above.

Isolation of RNA and reverse transcription
Cells were collected at 0, 0.5, 1, and 3 h after treatment, and total RNA was isolated with TRIzol reagent (Invitrogen, Milano, Italy), according to the manufacturer’s protocol. RNA was subjected to reverse transcription with random hexamers, as previously described (14).

Relative quantitation of cyclin D1 gene expression by real-time PCR
Relative quantitative PCR was performed to assess human cyclin D1 gene expression using the ABI Prism 7700 (Applera Italia, Monza, Italy) and assay on demand technology (www.appliedbiosystems.com, assay ID: Hs00233498; Applera Italia). Human 18S expression, considered as reference, was evaluated using previously described primers and probe (14). Ninety six-well optical plates with reaction mixture were heated for 2 min at 50 C, 10 min at 95 C, followed by 40 cycles of PCR, consisting of 15 sec at 95 C and 1 min at 60 C. At the end of the run, PCR products were analyzed by gel electrophoresis to confirm that a single product was amplified. The comparative CT method for relative quantitation of gene expression (user bulletin 2, Applied Biosystems) was used to determine cyclin D1 expression levels referring all sample vs. mRNA from cells starved for 48 h. Experiments were carried out in triplicate for each data point, and data analysis was performed by using Sequence Detection System 1.7 software (Applera, Italia).

Detection of nuclear Rb phosphorylation at Ser-780
Nuclear proteins were isolated as previously described (15). TT cells were collected at 0, 3, 6, 9, and 12 h after treatment and resuspended in 900 µl RBS buffer [10 mM Tris-HCl (pH 7.4), 10 mM NaCl, 3 mM MgCl2, 0.002% NaN3, 50 nM dithiothreitol (DTT), 0.2 µg/ml leupeptin, 1 µg/ml aprotinin, 100 nM phenylmethylsulfonyl fluoride (PMSF), 4 nM EGTA, and 100 nM sodium orthovanadate] and 100 µl of 5% Nonidet-P40. Cells were kept on ice for 5 min and centrifuged at 4 C for 5 min at 3300 x g. Pellets were then washed with 1 ml RBS buffer and centrifuged at 4 C for 5 min at 7400 x g. Pellets were resuspended in 60 µl buffer C [20 mM HEPES (pH 7.5), 0.42 M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA (pH 8.6), 25% glycerol, 0.01% NaN3, 0.5 mM DTT, 1 mM PMSF, 2 µg/ml leupeptin, 100 µg/ml aprotinin, and 1 mM sodium orthovanadate] and kept on ice for 40 min. Samples were centrifuged at 4 C for 10 min at 17,900 x g and 1 volume of buffer D [20 mM HEPES (pH 7.5), 50 mM KCl, 0.2 mM EDTA (pH 8.6), 20% glycerol, 0.01% NaN3, 0.5 mM DTT, 1 mM PMSF, 2 µg/ml leupeptin, 100 µg/ml aprotinin, and 1 mM sodium orthovanadate] was added to the supernatants. Nuclear protein cell extracts were measured by using the BCA protein assay reagent kit (Pierce Biotechnology), and equal amounts of nuclear proteins (~70 µg) were fractionated on 8% SDS-PAGE and transferred by electrophoresis to nitrocellulose membranes (Schleicher & Schuell). The membranes were incubated with polyclonal anti phospho-Rb (Ser-780) antibody (Cell Signaling Technology; source: rabbit; catalog no. 9307L) and antitotal Rb (C15) antibody (Santa Cruz Biotechnology, Santa Cruz CA; source: rabbit; catalog no. sc-50) used at 1:1000 and with 0.2 µg/ml antilamin A antibody (Sigma-Aldrich; source rabbit; L1293). Horseradish peroxidase-conjugated antirabbit IgG (Dako) was used at 1:2000 and binding was detected by SuperSignal West femto maximum sensitivity (Pierce).

Cyclin D1 turnover analysis in TT cells
TT cells were grown to 50% confluence, rinsed twice with PBS, and partially synchronized in G0/G1, starving the cells in serum-free medium for 48 h. Cells were then released for 2 h after medium renewal with 10% FBS. Cycloheximide 50 µg/ml was added to the cells together with or without SRIH and BIM-23120 10–8 M. Total protein cell extracts were fractionated on 10% SDS-PAGE, transferred by electrophoresis to nitrocellulose membranes, and blotted with the cyclin D1-specific antibody. Antibody complexes were visualized by enhanced chemiluminescence, and the cyclin D1 decay was determined by spot densitometry.

Statistical analysis
Results are expressed as the mean ± SD. A preliminary analysis was carried out to determine whether the data sets conformed to a normal distribution, and a computation of homogeneity of variance was performed using Bartlett’s test. The results were compared within each group and between groups using ANOVA. If the F values were significant (P < 0.05), Student’s paired or unpaired t test was used to evaluate individual differences between means. P < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
SRIH and BIM-23120 cause growth inhibition associated with reduction in [3H] thy incorporation and transient increase in G2/M phase
To verify the effects of SRIH and the SSTR2 selective agonist, BIM-23120, on TT cell proliferation and DNA synthesis, after a 48-h starvation, TT cells were incubated in F-12 supplemented with 10% FBS at different time points without or with SRIH or BIM-23120 at 10–8 M. As shown in Fig. 1AGo (top panel), SRIH and BIM-23120 significantly reduced cell viability after 48 h (–20 and –35% vs. control cells, respectively; P < 0.05) and 54 h (–16 and –22% vs. control cells, respectively; P < 0.05), whereas no significant effect was evident after 24, 36, and 60 h of treatment. As indicated in Fig. 1AGo (bottom panel), SRIH and BIM-23120 also induced a significant decrease in [3H] thy incorporation after 48 h (–38 and –35% vs. control cells, respectively; P < 0.05) and 54 h (–25 and –16% vs. control cells, respectively; P < 0.05). Cell-counting experiments showed that treatment with SRIH or BIM-23120 caused a significant decrease in cell number as compared with control cells at 48 h (–25 and –33% vs. control cells, respectively; P < 0.05) and 54 h (–20 and –25% vs. control cells, respectively; P < 0.05) (Fig. 2Go). Fluorescence-activated cell sorter analysis showed an 8% increase in S phase and a 20% decrease in G1 phase in cells released from starvation by incubation in F-12 supplemented with 10% FBS for 48 h. Moreover, a significant approximately 10% increase in G2/M phase (P < 0.01) and a approximately 7% reduction in S phase (P < 0.05) were detected in TT cells treated for 48 h with SRIH or BIM-23120, compared with control cells (Fig. 3Go). This effect was not observed for longer incubation times (data not shown). All these experiments are consistent with the demonstration that SSTR2 is indeed expressed in cycling TT cells (Fig. 1BGo).


Figure 1
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  		FIG. 1. Effect of SRIH and SSTR2-selective agonist on cell proliferation and [3H] thy incorporation in TT cells. Top, TT cells were seeded in 96-well plates, serum starved for 48 h, and incubated with F-12 supplemented with 10% FBS without or with 10–8 M SRIH or BIM-23120, a SSTR2 selective agonist. Control wells were treated with vehicle solution. Cell viability was determined at 24, 36, 48, 54, and 60 h. Data from six individual experiments were evaluated independently with eight replicates and expressed as the mean ± SD percent cell proliferation inhibition vs. untreated control cells. *, P < 0.05 vs. control. Bottom, TT cells were seeded in 24-well plates, serum starved for 48 h, and incubated with F-12 supplemented with 10% FBS, without or with 10–8 M SRIH or BIM-23120. [3H] thy incorporation was determined at 24, 36, 48, 54, and 60 h. Control wells were treated with vehicle solution. [3H] thy incorporation was measured as radioactivity in TCA-precipitated material. Data from six individual experiments were evaluated independently with four replicates and expressed as the mean ± SD percent [3H] thy incorporation inhibition vs. untreated control cells. *, P ≤ 0.05 vs. control. B, Total TT cell lysate (100 µg) was analyzed by Western blot for the expression of SSTR2 (~72 kDa). The immunoblot is representative of three individual experiments with similar results.

 

Figure 2
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  		FIG. 2. Effects of SRIH and BIM-23120 on TT cell number. TT cells were seeded in 24-well plates, serum starved for 48 h, and incubated with F-12 supplemented with 10% FBS, without or with 10–8 M SRIH or BIM-23120. Cell number was determined at 48, 54, and 60 h using a hemocytometer. Control wells were treated with vehicle solution. Data from four individual experiments were evaluated independently with six replicates. Data are expressed as mean ± SD. *, P ≤ 0.05 vs. control.

 

Figure 3
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  		FIG. 3. Effect of SRIH and BIM-23120 on cell cycle distribution. Subconfluent TT cells were serum starved for 48 h (0) and subsequently incubated with F-12 supplemented with 10% FBS without (control) or 10–8 M SRIH or BIM-23120 for 48 h. Cell cycle distribution was determined by flow cytometry in six individual experiments. Mean percentages of cells in G1 (white bars), S (black bars), or G2/M phase (gray bars) are presented as mean ± SD. *, P < 0.05 and **, P < 0.01 vs. control.

 
Effect of SRIH and BIM-23120 on cell cycle protein expression and GSK-3ß pathway in TT cells
G2/M to G1 phase progression depends on the coordinated expression of cyclins that bind to and activate Cdks. A reduction in cyclin D1 expression could result in an increase in the number of cells in the G2/M phase (16). Therefore, we investigated whether SSTR2-mediated cell cycle transient inhibition is associated with a modification in the expression of cell cycle proteins. After a 48-h starvation, cells were incubated with F-12 supplemented with 10% FBS without or with SRIH or BIM-23120 at 10–8 M for 1, 3, 6, 9, and 12 h, and Western blot analysis for cyclin D1, phospho-cyclin D1 Thr286, cyclin D2, Cdk4/6, and cyclin E was performed (Fig. 4Go). In control cells cyclin D1, cyclin D2, Cdk4/6, and cyclin E protein levels increased, compared with starved cells after 3, 6, 9, and 12 h of incubation. Treatment with SRIH determined a strong reduction in serum-induced cyclin D1 at 6 and 9 h and cyclin D2 at 9 h, with a reduction in Cdk4 and cyclin E at 3 h. Treatment with BIM-23120 reduced serum-induced cyclin D1 at 3, 6, and 9 h; cyclin D2 at 6 and 9 h; Cdk4 and cyclin E at 3 and 6 h; and Cdk6 at 3 h. These effects were not observed for longer incubation times.


Figure 4
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  		FIG. 4. Effect of SRIH and BIM-23120 on cell cycle regulatory proteins. TT cells were serum starved for 48 h (0) and then incubated with F-12 supplemented with 10% FBS without (control) or with 10–8 M SRIH or BIM-23120 for 1, 3, 6, 9, and 12 h. One hundred milligrams of total protein extracts were analyzed by Western blot for the expression of cyclin D1 (36 kDa), phosphocyclin D1 Thr286, cyclin D2 (33–35 kDa), Cdk4 (33 kDa), Cdk6 (40 kDa), cyclin E (50 kDa), and actin (42 kDa) (A). The immunoblot is representative of six individual experiments with similar results, in which each band was evaluated quantitatively and each protein to actin ratio was expressed as mean ± SD (B).

 
To verify whether the observed reduction in cyclin D1 protein level correlates with changes in cyclin D1 gene expression, we performed real-time quantitative PCR on cDNA from TT cells incubated for 0.5, 1, and 3 h with F-12 supplemented with 10% FBS without or with SRIH or BIM-23120 at 10–8 M. As shown in Fig. 5Go, in control cells cyclin D1 mRNA level significantly increased at all time points [1.4-, 1.9-, and 2.4-fold vs. starved cells at 0.5 (P < 0.05), 1 (P < 0.01), and 3 h (P < 0.01), respectively]. Treatment with SRIH or BIM-23120 did not modify serum-induced cyclin D1 mRNA level at any time point. We also performed time-course studies showing that cyclin D1 mRNA levels under SRIH or BIM-23120 treatment were comparable with those recorded in the control cells also from 3 to 24 h (data not shown).


Figure 5
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  		FIG. 5. Effect of SRIH and BIM-23120 on cyclin D1 gene expression. TT cells were serum starved for 48 h (0) and then incubated with F-12 supplemented with 10% FBS without (control) or with 10–8 M SRIH or SSTR2 selective agonist, BIM-23120, for 0.5, 1, and 3 h. Relative cyclin D1 mRNA mean levels (fold induction ± SD) in treated cells vs. starved cells were evaluated by real-time PCR in six individual experiments. *, P < 0.05 and **, P < 0.01 vs. starved cells (0).

 
Because we failed to detect any change in mRNA expression, we investigated whether the observed reduction in cyclin D1 protein level could be due to proteolysis. It is well known that GSK-3ß phosphorylates cyclin D1 at Thr286 (17, 18), triggering cyclin D1 turnover by the ubiquitin pathway (17, 19). We therefore evaluated cyclin D1 Thr286 phosphorylation level as well as GSK-3ß activation under treatment with SRIH or the SSTR2 selective agonist. As shown in Fig. 4Go, in control cells phosphocyclin D1 level increased up to 3 h and remained stable up to 12 h. In parallel, treatment with SRIH further increased phosphocyclin D1 level after 6 and 9 h, compared with control cells. Moreover, already after 1 h, BIM-23120 increased phosphocyclin D1 level, which remained elevated up to 9 h, compared with control cells. Consequently, we investigated GSK-3ß activation under the same conditions. It has been previously shown that phosphorylation at Ser9 inhibits GSK-3ß activity (20). As shown in Fig. 6Go, in control cells GSK-3ß Ser9 phosphorylation increased after 1 h and remained stable up to 12 h, suggesting that GSK-3ß activity is restrained. In contrast, treatment with SRIH decreased GSK-3ß Ser9 phosphorylation after 9 and 12 h. In cells treated with BIM-23120, GSK-3ß Ser9 phosphorylation level was reduced as soon as after 1 h and persisted low up to 9 h. These results are consistent with GSK-3ß activation under treatment with SRIH or the SSTR2 selective agonist.


Figure 6
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  		FIG. 6. Effect of SRIH and BIM-23120 on Ser9 GSK-3ß phosphorylation level. TT cells were serum starved for 48 h (0) and then incubated with F-12 supplemented with 10% FBS without (control) or with 10–8 M SRIH or BIM-23120, the SSTR2 selective agonist, for 1, 3, 6, 9, and 12 h. Total protein extracts were assayed for phospho Ser9 GSK-3ß and total GSK-3ß by immunoblot. Each lane was loaded with 100 µg protein. The immunoblot is representative of three individual experiments with similar results, in which each band was evaluated quantitatively, and phospho-Ser9 GSK-3ß to total Gsk-3ß ratio was expressed as mean ± SD.

 
Effect of SRIH and BIM-23120 on cyclin D1 turnover in TT cells
To determine whether SRIH and BIM-23120 treatment induces a decline in the steady-state cyclin D1 protein levels due to increased protein degradation, we determined cyclin D1 protein turnover using cycloheximide blocking experiments in the TT cell line. Consistent with a previous study (21), in the control samples (Fig. 7Go), cyclin D1 protein half-life was approximately 60 min. Cyclin D1 protein half-life from cells treated with SRIH or with BIM-23120 was nearly 45 and 30 min, respectively (Fig. 7Go), suggesting that these compounds induce cyclin D1 protein degradation.


Figure 7
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  		FIG. 7. Effect of SRIH and BIM-23120 on cyclin D1 turnover. TT cells were serum starved for 48 h (0) and then incubated with F-12 supplemented with 10% FBS. After 2 h, cells were incubated without (control) or with 10–8 M SRIH or BIM-23120 in the absence or presence of cycloheximide 50 mg/ml for 0, 5, 20, 30, 40, 60, 120, and 180 min (A). The relative intensity was determined by spot densitometry and represented as percent cyclin D1 decline vs. control (B). The results represent the mean ± SD of three independent experiments.

 
Effect of SRIH and BIM-23120 on Ser-780 Rb phosphorylation in TT cells
To verify whether the observed reduction in cyclin D1 protein might influence endogenous Rb phosphorylation level, we performed Western blot analysis for Ser-780 Rb and total Rb levels with nuclear extracts from TT cells incubated for 3, 6, 9, and 12 h with F-12 supplemented with 10% FBS without or with SRIH or BIM-23120 at 10–8 M. As shown in Fig. 8Go, phosphorylation of Rb on Ser-780, a target for specific cdk4/6-dependent phosphorylation in vitro, significantly increased in control cells up to 9 h of incubation (P < 0.05 at 3 and 6 h, and P < 0.01 at 9 h vs. starved cells) and then decreased at 12 h. Treatment with SRIH significantly reduced serum-induced Ser-780 Rb phosphorylation at 6 and 9 h (P < 0.05 vs. control). Treatment with BIM-23120 determined a stronger reduction in serum-induced Ser-780 Rb phosphorylation from 3 up to 9 h (P < 0.05 vs. control). Total Rb protein level was constant throughout the experiment in all samples.


Figure 8
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  		FIG. 8. Effect of SRIH and BIM-23120 on Ser 780 Rb phosphorylation level. TT cells were serum starved for 48 h (0) and then incubated with F-12 supplemented with 10% FBS without (control) or with 10–8 M SRIH or BIM-23120 for 3, 6, 9, and 12 h. Total nuclear protein extracts were assayed for phospho Ser-780 Rb (Ser-780 Rb) and total Rb (total Rb) by immunoblot. Each lane was loaded with 70 µg protein. The immunoblot is representative of six individual experiments, in which each band was evaluated quantitatively, and Ser-780 Rb to total Rb ratio was expressed as mean ± SD. *, P < 0.05 vs. control cells; #, P < 0.05, and ##, P < 0.01 vs. starved cells (0).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The antiproliferative effect of SRIH has been demonstrated in many biological systems, and its mechanism of action has been thoroughly investigated, providing different, and sometimes conflicting results depending on the experimental model. It has been demonstrated that the growth-inhibitory action of SRIH can be mediated by tyrosine phosphatase activation (9, 22, 23) and induction of p27, a cyclin kinase inhibitor known to block cell cycle progression by targeting G1 Cdks (24, 25). Furthermore, there is evidence showing that SRIH inhibits Cdk2 activity (24, 26), suggesting that SRIH might exert its antiproliferative effects mainly by modulating G1 phase protein expression and function.

We previously demonstrated that SRIH inhibits TT cell proliferation mainly by interacting with SSTR2 (7, 27), which, in turn, activates SHP-1 (9). In this study, we investigated the effects of SRIH and a SSTR2 selective agonist on cell cycle progression and cell cycle proteins in TT cells.

Our results confirm previously reported data, showing that SRIH and the SSTR2 selective agonist inhibit TT cell proliferation and DNA synthesis, and demonstrate that these compounds induce a transient accumulation of TT cells in G2/M phase. This finding is in line with previous evidence, showing that in MCF7 human mammary tumor cells, a SRIH analog interacting with SSTR2 induces a G2/M transient accumulation (28). Moreover, our data show a reduction in cyclin D1 protein expression, together with a minor reduction in cyclin D2 protein, a member of the cyclin D family, involved in cell cycle progression. These data would suggest that cyclin D2 has a minor role in the control of TT cell cycle, in agreement with previous evidence, indicating that endogenous cyclin D2 protein cannot compensate normal transit through the cell cycle in the absence of cyclin D1 (16). However, previous studies indicated that SRIH analog treatment of SSTR2 transfected CHO cells induces G1 arrest (24). On the other hand, a reduction or loss of cyclin D1 expression in cells undergoing transient accumulation in G2/M phase has been shown (29, 30). Moreover, our previous studies showed that SRIH activates SHP-1 and down-regulates the MAPK pathway in TT cells (9). It has been shown that MAPK pathway positively regulates cyclin D1 expression (10). However, the latter studies have been performed in the Chinese hamster lung fibroblast cell line CCL39, whereas our experiments, carried out in the human MTC cell line TT, did not show any change in cyclin D1 mRNA levels up to 24 h. Because the interaction of various transcription factors is tissue specific, we can hypothesize that the MAPK pathway might have different target proteins in the two cell lines.

Taken together these findings suggest that cyclin D1 might be involved in the regulation of transition from G2/M to G1 phase, triggering specific checkpoints in G2/M phase and enabling the cell to enter G1 normally. Nevertheless, our data do not rule out an important influence of cyclin D1 in early G1 phase, but they point out a wider range of actions for cyclin D1 on cell cycle regulation.

Concerning cyclin D1-associated kinases, it has been previously demonstrated that cells undergoing G2 delay display an inhibition of cyclin D-Cdk4 activity (31). Accordingly, our data show that under treatment with both SRIH and the SSTR2 selective agonist G2/M accumulation associates with a decrease in Cdk4 protein levels, likely resulting in a decreased activity of the cyclin D1-Cdk4 complex in TT cells. Indeed, it is well documented that the cyclin D-Cdk4/6 complex specifically phosphorylates RB protein at Ser-780 (32, 33). Our results show a reduction in Ser-780 Rb phosphorylation levels in parallel with a decrease in Cdk4 protein levels. Furthermore, there is evidence indicating that Rb phosphorylation by cyclin D-Cdk4/6 complex is required for subsequent Rb phosphorylation by cyclin E/Cdk2 (34), thus promoting progression into the cell cycle (35). In agreement with Pages et al. (24), we observed a reduction in cyclin E protein levels under treatment with SRIH and a SSTR2 selective agonist. The inhibitory effects of these compounds on cell cycle progression might be therefore mediated also by a reduction in cyclin E, even if an involvement of cyclin E in G2/M accumulation has never been demonstrated, so far. Moreover, our results show that SRIH and its SSTR2 agonist do not influence cyclin D1 mRNA levels, suggesting that these compounds might act at posttrascriptional level. It has been demonstrated that cyclin D1 protein degradation is mediated by Thr286 phosphorylation-triggered, ubiquitin-dependent proteolysis (17).

Our data show that cyclin D1 phosphorylation at Thr286 increases in control cells over time, in parallel with the increase in total cyclin D1 protein levels. The demonstration of a further increase in cyclin D1 Thr286 phosphorylation under treatment with both SRIH and the SSTR2 selective agonist supports the hypothesis that these compounds promote the degradation of cyclin D1 protein. This hypothesis is further supported by the evidence that cyclin D1 protein half-life is reduced under treatment with SRIH or the SSTR2 selective agonist. Cyclin D1 protein degradation is induced by GSK-3ß, which specifically phosphorylates cyclin D1 at Thr286, triggering a rapid cyclin D1 turnover (18). GSK-3ß activity is negatively controlled by a pathway that sequentially involves Ras, phosphatidylinositol-3-OH kinase, and Akt signaling (20), which, in turn, are down-regulated by SRIH (26, 36). Indeed, our results show that treatment with both SRIH and the SSTR2 selective agonist reduce GSK-3ß Ser9 phosphorylation levels, reflecting the induction in GSK-3ß activity (20). Whereas SSTR2 selective agonist early activates GSK-3ß, the effect of SRIH on GSK-3ß phosphorylation is delayed (9 h). However, cyclin D1 phosphorylation under SRIH treatment is apparent at earlier incubation times, suggesting that SRIH might exert this effect also by activating pathways different from GSK-3ß. On the other hand, the observed effects might also be due to the possible activation of other SSTR subtypes by the multiligand agonist SRIH. Indeed, SSTR subtypes different from SSTR2 might transduce different effects on cell cycle proteins.

In conclusion, this study demonstrates that activation of SSTR2 inhibits TT cell growth mainly by decreasing serum-induced cyclin D1 protein level, thus resulting in a reduced Ser-780 Rb phosphorylation and a delay in G2/M phase. Our data further underline the importance of SSTR2 in mediating the antiproliferative effects of SRIH, indicating a direct and strong effect on cyclin D1-cdk4 complex. However, our results do not rule out a possible role for other mechanisms in mediating SSTR2-induced effects on cell proliferation.


   Footnotes
 
This work was supported by grants from the Italian Ministry of University and Scientific and Technological Research (University of Ferrara: 60%–2005 and MIUR 2005 060 839-004), IPSEN, Fondazione Cassa di Risparmio di Ferrara, and the Associazione Ferrarese dell’Ipertensione Arteriosa to the University of Ferrara.

F.T., M.C.Z., A.B., D.P., A.L., and E.C.d.U. have nothing to declare. M.D.C. is employed by Biomeasure Inc./IPSEN.

First Published Online April 6, 2006

Abbreviations: DTT, Dithiothreitol; FBS, fetal bovine serum; GSK, glycogen synthase kinase; [3H] thy, [3H]thymidine; MTC, medullary thyroid carcinoma; PI, propidium iodide; PMSF, phenylmethylsulfonyl fluoride; SHP, Src homology-2-containing protein; SRIH, somatostatin; SSTR, SRIH receptor subtypes.

Received November 22, 2005.

Accepted for publication March 28, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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