Abstract
Ligand-activated somatostatin receptors (SSTRs) initiate cytotoxic or cytostatic antiproliferative signals. We have previously shown that cytotoxicity leading to apoptosis was signaled solely via human (h) SSTR subtype 3, whereas the other four hSSTR subtypes initiated a cytostatic response that led to growth inhibition. In the present study we characterized the antiproliferative signaling mediated by hSSTR subtypes 1, 2, 4, and 5 in CHO-K1 cells. We report here that cytostatic signaling via these subtypes results in induction of the retinoblastoma protein Rb and G1 cell cycle arrest. Immunoblot analysis revealed an increase in hypophosphorylated form of Rb in agonist-treated cells. The relative efficacy of these receptors to initiate cytostatic signaling was hSSTR5>hSSTR2>hSSTR4∼hSSTR1. Cytostatic signaling via hSSTR5 also induced a marginal increase in cyclin-dependent kinase inhibitor p21. hSSTR5-initiated cytostatic signaling was G protein dependent and protein tyrosine phosphatase (PTP) mediated. Octreotide treatment induced a translocation of cytosolic PTP to the membrane, whereas it did not stimulate PTP activity when added directly to the cell membranes. C-tail truncation mutants of hSSTR5 displayed progressive loss of antiproliferative signaling proportional to the length of deletion, as reflected by the marked decrease in the effects of octreotide on membrane translocation of cytosolic PTP, and induction of Rb and G1 arrest. These data demonstrate that the C-terminal domain of hSSTR5 is required for cytostatic signaling that is PTP dependent and leads to induction of hypophosphorylated Rb and G1 arrest.
INTRODUCTION
The antiproliferative actions of somatostatin (SST) signaled via cell surface SST receptors (SSTRs) regulate cellular protein phosphorylation and elicit cytostatic (growth arrest) and cytotoxic (apoptosis) responses in tumor cells. For instance, SST treatment causes apoptosis in MCF-7 and AtT-20 cells, whereas it induces cell cycle arrest in GH3 cells (1–5). Such discrepant findings may be due to the existence of five distinct SSTR subtypes and their differential expression in these tumor cells (6–10). We have reported that human (h) SSTR3 is the only subtype that is capable of cytotoxic signaling: upon ligand activation, cells transfected with hSSTR3 respond with induction of wild-type (wt) tumor suppressor protein p53, the proapoptotic protein Bax, and an acidic endonuclease and intracellular acidification and undergo apoptosis (11, 12). The antiproliferative action of SST is also signaled via SSTRs 2, 4, and 5. However, neither apoptosis nor changes in any of the above parameters were seen in cell lines stably expressing these subtypes (11, 13). While SST was shown to inhibit cell growth via hSSTRs 2, 4, and 5, such a conclusion was based on measurement of thymidine incorporation or cell number at a single time point during SST treatment (14–17). Thus, the mechanism underlying the antiproliferative signaling mediated by the SSTR subtypes incapable of triggering apoptosis remained unknown. Since these SSTR subtypes do not initiate apoptotic signals, it appeared likely that they may transduce cytostatic signals leading to cell cycle arrest.
Cytostatic events leading to G1 cell cycle arrest are associated with the induction of two proteins Rb (retinoblastoma tumor suppressor protein) and p21 (cyclin-dependent kinase inhibitor, also called Waf-1/Cip1) (18). Rb is a phosphorylated protein: it remains hyperphosphorylated (ppRb) in S and G2/M phases and becomes hypophosphorylated (pRb) in G1. pRb negatively regulates the G1/S transition and promotes accumulation of cells in the G1 phase (18, 19). Rapid phosphorylation of Rb occurs before entry of cells into S phase. While Rb functions independently of p53, p21 mediates p53-dependent G1 arrest (18, 20). Nevertheless, overexpression of p21 can induce G1 arrest in the absence of p53 induction (21). To determine whether the antiproliferative signaling via these hSSTRs causes cell cycle arrest and to identify the molecular mediators involved in this process, we evaluated the effect of SST in CHO-K1 cells expressing hSSTRs 1, 2, 4, and 5 on cell cycle progression and induction of Rb and p21. We report here that SST-induced G1 cell cycle arrest in these cells is due mainly to the induction of Rb. Maximal effect was exerted via hSSTR5 followed by hSSTR 2, 4, and 1. In hSSTR5-expressing cells, a major portion of SST-induced Rb was hypophosphorylated. SST-induced G1 arrest and induction of Rb were pertussis toxin sensitive, G protein dependent, and protein tyrosine phosphatase (PTP) dependent. In octreotide (OCT)-treated cells there was a redistribution of PTP activity from the cytosol to the membrane. Mutational analysis of the C tail of this receptor revealed that the C tail of the receptor is essential for PTP-dependent cytostatic signaling.
RESULTS
We first compared the effect of SST agonists [OCT (hSSTRs 2, 3, and 5) or d-Trp8 SST-14 (hSSTRs 1 and 4)] on cell cycle parameters in CHO-K1 cells expressing individual hSSTRs. Cells were incubated for 24 h in the absence or presence of agonists at a maximal stimulatory concentration of 100 nm. (11). Cell cycle analysis revealed that cells expressing hSSTRs 1, 2, 4, and 5 responded with a decrease in the rate of proliferation. This was evident from the agonist-induced increase in cells in G1 and a decrease in S (Fig. 1). The greatest cytostatic response was elicited through hSSTR 5 followed by hSSTR2>hSSTR4∼hSSTR1. The effect of agonist treatment on cell cycle parameters is shown in Fig. 2. In addition to the changes in G1 and S phases, a relative increase of cell number in G2/M was also seen. The absence of oligonucleosomal DNA fragmentation, even after treatment for 48 h, indicated that SST-induced cell cycle arrest via hSSTR5 did not lead to apoptosis (data not shown).
Effect of Peptide Treatment on Cell Cycle Parameters in CHO-K1 Cells Expressing hSSTR1–5 Representative plots showing the phase distribution of cells incubated for 24 h in the absence (top panel) or presence (bottom panel) of 100 nm OCT (hSSTRs 2, 3, and 5) or d-Trp8 SST-14 (hSSTRs 1 and 4). Cellular DNA was stained with PI and analyzed by flow cytometry. An increase in G1 peak can be seen in cells expressing four of the five hSSTR subtypes (hSSTR5>hSSTR2>hSSTR4∼hSSTR1). This contrasts with the decrease in G1 peak and the appearance of a hypodiploid peak in the region A0 after peptide treatment in cells expressing hSSTR3.
Effect of Agonist Treatment on Cell Cycle Parameters in CHO-K1 Cells Expressing Individual hSSTR Subtypes The distribution of cells in G0/G1 (top panel), S (middle panel), and G2/M (bottom panel) were quantitated by analysis of PI-stained cells by flow cytometry. The increase in the number of cells G0/G1 was accompanied by a decrease in cell number in S phase. A small increase in G2/M was also evident in agonist-treated cells (mean ± se, n = 3). *, P < 0.005; **, P < 0.05.
Inhibition of cell cycle progression signaled via hSSTR 5 was associated with induction of Rb. The increase in intensity of fluorescence of immunolabeled Rb counterstained with fluorescein isothiocyanate (FITC)-conjugated second antibody was seen in all phases of the cell cycle after OCT treatment. Dual label analysis of fluorescence emissions of propidium iodide (PI) and immunostained Rb revealed that the majority of cells were in G0/G1 phase (Fig. 3A). Immunoblot analysis of cell extracts revealed that the level of Rb was low in untreated cells and was present mainly as ppRb. An OCT-induced increase in Rb was reflected in both hyper- and hypophosphorylated (ppRb and pRb) forms detectable by their differential electrophoretic mobility (Fig. 3B). A marked enlargement of the nuclei in hSSTR5-expressing cells was observed in OCT-treated cells (Fig. 3C), a typical characteristic of G1 arrested cells (22). OCT-induced increase in Rb was time dependent and was detectable by 4 h (2.7 ± 0.9 fold) and was maximal at 24 h (8.1 ± 0.8 fold) (Fig. 4A). Induction of Rb preceded the onset of G1 arrest since an increase in G1/S ratio, which is an index of inhibition of cell proliferation, was detectable only by 8 h (Fig. 4B). The ability of OCT to induce Rb during 24 h incubation was dose dependent and occurred over the concentration range 10–100 nm (Fig. 5A). Immunoblot analysis revealed a dose-dependent increase in both ppRb and pRb in OCT-treated cells (Fig. 5B).
hSSTR5-Mediated Antiproliferative Signaling in CHO-K1 Cells Causes G1 Cell Cycle Arrest Associated with Induction of Rb A, Flow cytometric analysis of hSSTR5 expressing cells incubated in the absence and presence of 100 nm OCT for 24 h. Scattergram represents dual label plot of FITC fluorescence of immunostained Rb measured on a log scale against PI fluorescence measured on a linear scale. In addition, PI fluorescence depicting the cell cycle distribution is shown on the top, and the FITC fluorescence of immunostained Rb is shown on the right of the scattergram. An OCT-induced increase in Rb occurred in all phases of the cell cycle and was associated with an increase in the number of cells in G1. Data are representative of three separate experiments. B, Western blot analysis of Rb in CHO-K1 cells. In addition to the total increase in Rb in OCT-treated cells, a significant portion was in the hypophosphorylated form (pRb) that could be distinguished from the hyperphosphorylated form (ppRb) on the basis of differential electrophoretic mobility. C, Nuclear morphology of PI-stained cells revealed nuclear enlargement, a feature that is characteristic of G1 arrested cells.
Time Dependency of hSSTR5-Mediated Induction of Rb and Inhibition of Cell Cycle Progression After incubation in the presence of 100 nm OCT for the indicated time, Rb and DNA were quantitated in the same cell populations by dual label flow cytometry (mean ± se, n = 3; *, P < 0.05; **, P<0.005). A, Rb was quantitated by flow cytometry after immunolabeling. Values represent as percent change in fluorescence intensity measured on a log scale and compared with that in untreated cells taken as 100% (mean ± se, n = 3). B, Inhibition of cell proliferation by OCT is reflected in the increase in the ratio of cells in G1 and S phases is evident by 8 h and was maximal at 24 h.
Dose-Dependent Induction of Rb by OCT via hSSTR5 A, Rb was measured by flow cytometry after immunolabeling in cells incubated with the indicated concentrations of the peptide for 24 h. Values represent percent change in fluorescence intensity measured on a log scale and compared with that in untreated cells taken as 100% (mean ± se, n = 3). B, Immunoblot demonstrating that OCT-induced augmentation in Rb is associated with a dose-dependent increase in hypophosphorylated form of Rb (pRb).
OCT-treated hSSTR5 cells also displayed an increase in p21 (3 ± 0.6 fold over basal level) and was of much smaller magnitude compared with that of Rb. OCT-induced increase in Rb and p21 was abolished by pertussis toxin pretreatment (Fig. 6). Sodium orthovanadate, an inhibitor of PTP, also abrogated the inductive effect of OCT on Rb and p21Waf1 in these cells. To confirm that PTP activity is involved in the cytostatic signaling via hSSTR5, we measured PTP activity in extracts of cells before and after incubation with SST. In OCT-treated cells there was a 40% increase in membrane-associated PTP while the cytosolic enzyme activity decreased by 20% (Fig. 7). By contrast, when added to the membrane fractions at the time of enzyme assay, OCT failed to stimulate PTP activity (data not shown). While the maximal induction of Rb was hSSTR5 mediated, three other subtypes were also found to initiate cytostatic signals leading to Rb induction (Fig. 8). The rank order potency of these SSTRs for signaling the increase in Rb was hSSTR5>2 >4>1, the same as that observed for triggering G1 arrest (Figs. 1 and 2). By contrast, no increase in Rb occurred in hSSTR3 expressing cells, in agreement with our previously reported finding that OCT does not induce G1 arrest via this subtype (11, 12).
hSSTR5-Mediated Cytostatic Signaling Is Pertussis Toxin Sensitive, G Protein Mediated, and PTP Dependent OCT-treated cells displayed an increase in p21 in addition to Rb. Fluorescence intensity of antibody-labeled Rb and p21 was quantitated by flow cytometry after immunolabeling. Values represent percent change in fluorescence intensity in OCT-treated cells measured on a log scale and compared with that in untreated cells taken as 100%. The increase in p21 in cells incubated with 100 nm OCT was less than that of Rb (3.0 ± 0.8 vs. 8.1 ± 0.8 fold, respectively). hSSTR5-signaled induction of these proteins was abolished by pretreatment of the cells with 100 ng pertussis toxin for 18 h before incubation with the peptide. Na orthovanadate (10 mg/ml) present during the incubation with the peptide also inhibited the action of OCT (mean ± se, n = 3, *, P < 0.005; **, P < 0.05).
Effect of OCT on PTP Activity in CHO-K1 Cells Expressing hSSTR5 PTP activity was measured in membrane and cytosolic fractions prepared from cells incubated in the absence or presence of 100 nm OCT for 24 h. The enzyme activity was measured using pNPP as the substrate (mean ± se, n = 3). By contrast, OCT did not stimulate PTP activity of the membrane fractions when added at the time of enzyme assay (not shown).
hSSTR Subtype Selectivity for Induction of Rb Maximum induction of Rb was seen in cells expressing hSSTR5, followed by hSSTR2>hSSTR4>hSSTR1. The fluorescence intensity of immunostained Rb was measured in cells incubated with 100 nm OCT (hSSTRs 2, 3, and 5) or d-Trp8 SST-14 (hSSTRs 1 and 4) for 24 h. Induction of Rb by agonists was quantitated by flow cytometry after immunolabeling. Values represent percent change in fluorescence intensity measured on a log scale and compared with that in untreated cells taken as 100% (mean ± se, n= 3; *, P < 0.05; **, P < 0.005).
The C tail of several G protein-coupled receptors has been implicated in G protein interaction and effector coupling (23, 24). To evaluate the importance of the C tail of hSSTR5 in cytostatic signaling, we investigated the effect of mutant hSSTR5 receptors with progressive truncation of the C tail (Fig. 9). These mutants have been previously reported to display binding characteristics and G protein coupling comparable to wild-type hSSTR5 (25). Progressive truncation of the C tail of hSSTR5 was associated with an impaired ability of OCT to signal activation of Rb and induce G1. Compared with the wild-type receptor, which triggered 8.1 ± 0.8 fold increase in Rb in response to OCT, the Δ347 mutant displayed only a 6.1 ± 0.4 fold increase in Rb (Fig. 10). The Δ338, Δ328, and Δ318 mutants displayed more marked loss in the ability to activate Rb in response to OCT (3.0 ± 0.9 fold for Δ338, 2.1 ± 0.7 fold for Δ328, and 1.3 ± 0.2 fold for Δ318). To determine whether progressive loss of ability to induce Rb parallels the decrease in membrane-associated PTP, we compared PTP activity in cytosolic and membrane fractions in cells incubated in the absence and presence of OCT. In contrast to the more than 2-fold increase induced by OCT pretreatment in cells expressing hSSTR5, only ∼25% increase occurred with the mutant Δ347, and no change was seen with the shorter hSSTR5 mutants (Fig. 11). Interestingly, the basal membrane-associated PTP activity was higher in untreated cells expressing each of the mutant receptors compared with wild-type hSSTR5.
Topographical Arrangement of Primary Amino Acid Sequence of hSSTR5 Showing the N-glycosylation Sites (CHO), S/T Phosphorylation Sites (•), and Palmitoylation Site (
) C tail truncation mutants of this receptor were generated by inserting stop codons at sites indicated by solid lines.
Effect of C Tail Deletion Mutations on hSSTR5-Initiated Rb Induction Induction of Rb by agonists was quantitated by flow cytometry after immunolabeling. Values represent percent change in fluorescence intensity measured on a log scale and compared with that in untreated cells taken as 100% (mean ± se, n = 3, *, P < 0.01; **, P < 0.0001).
Effect of C Tail Truncation on hSSTR5-Signaled Change in Cellular Distribution of PTP Activity in CHO-K1 Cells PTP activity was measured using pNPP as the substrate in membrane and cytosolic fractions of cells incubated for 2 h in the absence and presence of 100 nm OCT (mean ± se, n= 3; *, P < 0.0001; **, P < 0.005).
DISCUSSION
The present study establishes that SSTR-mediated antiproliferative signaling elicits subtype-selective cytostatic effect via hSSTRs 1, 2, 4, and 5. In CHO-K1 cells expressing each of these four hSSTR subtypes, there was decreased proliferation due, in part, to a G1 cell cycle arrest associated with an increase in Rb. The extent of Rb induction and inhibition of cell cycle progression was the greatest in CHO-K1 cells expressing hSSTR5, followed by hSSTR2>hSSTR4∼hSSTR1. A significant proportion of Rb induced via hSSTR5 was present in a hypophosphorylated form as evident from its greater electrophoretic mobility. We show that OCT treatment caused nuclear enlargement in hSSTR5-expressing cells, a feature that is characteristic of cells in G1 arrest (22). Additionally, hSSTR5-mediated cytostatic signaling did not lead to apoptosis. While the cytostatic action exerted through hSSTR5 by OCT was dose- and time dependent, such an effect occurred with a relatively slow time course and could be seen only at concentrations greater than 10 nm. This is in contrast to the greater sensitivity of hSSTR3-mediated induction of wild-type p53, which was clearly discernible within minutes and could be elicited at less than 10 nm concentration of OCT (11).
Pretreatment of cells with PTx abolished the induction of Rb, p21, and G1 arrest, indicating that the cytostatic signaling by hSSTRs 1, 2, 4, and 5 is G protein dependent. Likewise, our finding that orthovanadate abolishes the effects of SST suggests a mediatory role for PTP in the cytostatic signaling initiated via hSSTRs 1, 2, and 4 as well. PTP-mediated antimitogenic effect of SST has previously been reported to be signaled through hSSTR1, mouse and human SSTR2, human and mouse SSTR3, and rat SSTR4 (11, 15, 16, 26–28). By contrast, rat SSTR5-initiated antiproliferative signaling was found to be PTP independent (14, 17). The present findings suggest that the antiproliferative signaling via hSSTR 5 leading to growth inhibition is also PTP dependent and contradicts the reported inability of the rat homolog of SSTR5 to regulate PTP (14). Such a difference between the rat and human SSTR5 receptors is surprising given the high degree of C-terminal sequence identity between the two receptors. It remains to be seen whether structural differences in other regions of rat and human SSTR5 contribute to their divergent behavior.
The mechanism involved in SST induction of hypophosphorylated Rb remains to be elucidated. The concomitant, albeit smaller, induction of p21 raises the possibility that it may inhibit cyclin-dependent kinase-mediated phosphorylation of Rb that is required for the cells to exit G1. Alternatively, SST may activate phosphatase(s) that may dephosphorylate hyperphosphorylated Rb. Evidence for the existence of such a phosphatase comes from studies using anticancer drugs that promote p53-independent G1 arrest in the absence of p21 induction (29). It remains to be tested whether hSSTR5- mediated increase in hypophosphorylated Rb is due to activation of Rb phosphatase alone or in conjunction with p21-mediated inhibition of Rb phosphorylation. Another possibility is that SST may inhibit Ca2+/calmodulin-mediated hyperphosphorylation of Rb (30, 31). Cross-talk between SST-induced PTP and mitogenic signaling pathways involving mitogen-activated protein (MAP) kinase may also contribute to the regulation of serine phosphorylation in Rb as well as cell cycle progression. It has been shown that cell cycle progression due to induction of cyclin-dependent kinase and phosphorylation of Rb can occur after MAP kinase activation (32). It is likely that inhibition of MAP kinase activity by SST may be an additional factor involved in its cytostatic signaling. SSTR regulation of MAP kinase activation is complex and involves inhibition by SSTR2 and SSTR5, stimulation through SSTR4, or a transient increase followed by subsequent decrease elicited by (murine) SSTR3 (33). Gβγ-subunit-mediated activation of Ras is implicated in the induction of MAP kinase (34, 35). On the other hand, PTP-dependent regulation of serine/threonine phosphorylation inactivates Raf-1, which functions downstream of Ras in the mitogenic signaling cascade (27, 36–39). Thus, regulation of MAP kinase cascade by SST may occur at two levels: activation of Ras by βγ-subunits of G protein and tyrosine phosphorylation-dependent inactivation of MAP kinase. Thus, it is plausible that activation of different phosphorylation/dephosphorylation mechanisms by SST elicited in a receptor subtype-specific or cell-specific manner may exert dual effects on cell growth and proliferation (40, 41). A direct correlation between subtype-selective change(s) in MAP kinase and cell cycle arrest or apoptosis remains to be established.
Of the five hSSTRs, only hSSTR3 induces cytotoxicity, the other four subtypes being cytostatic (Refs. 11, 13 and the present study). We have previously reported that the Δ347, Δ338, Δ328, and Δ318 hSSTR5 mutants show progressive loss of the ability to inhibit forskolin-stimulated cAMP and variable impairment of agonist-dependent desensitization and internalization responses (25). This suggests a multifunctional role of the C tail of hSSTR5 in mediating effector coupling, desensitization, and internalization (25). Here we have extended an analysis of these mutants to their ability to regulate membrane-associated tyrosine phosphatase activity and to determine whether decreased potency to recruit cytosolic PTP to the membrane may account for their inability to initiate cytostatic signaling. After OCT treatment, only a 25% increase in PTP activity in the membrane fraction was seen in cells expressing hSSTR5Δ347, in contrast to the 100% increase detected in cells expressing the wild-type receptor. Membrane-associated PTP activity did not increase in response to OCT in cells expressing Δ338, Δ328, and Δ318 mutants. Surprisingly, however, the enzyme activity was 20–35% higher in the membrane fraction of these cells under basal conditions than in hSSTR5-expressing cells. We do not know the reason for this, but this observation raises the intriguing possibility that, in the absence of ligand activation, the C tail of the wild-type receptor may inhibit the association of PTP with the membrane. Such a phenomenon has not previously been described. However, chronic association of the tyrosine phosphatase SHP-1 to the killer cell- inhibitory receptor in natural killer cells has been reported to tonically inhibit the function of the receptor in the inactivated state; dissociation of SHP-1 upon receptor ligation restores its function (42). Despite its inability to recruit cytosolic PTP to the membrane in cells expressing these mutants, OCT was still capable of inducing Rb, albeit with progressively less efficiency paralleling the length of C tail deletion. While this raises the possibility that OCT may be able to elicit cytostatic signaling through the PTP already present at the membrane, alternate, PTP-independent mechanisms may also contribute to hSSTR5-initiated antiproliferative signaling. For instance, hSSTR5 can decrease intracellular Ca2+, thereby inhibiting cell growth (14, 43). While the nature of SST-induced, Ca2+-sensitive growth inhibition was not established in these studies, it may invoke hypophosphorylation of Rb. Indeed, Ca2+/calmodulin-dependent Rb hyperphosphorylation occurs during cell proliferation (30, 31).
In summary, the present findings demonstrate that SST peptides exert a cytostatic action via SSTR 1, 2, 4, and 5. Such subtype-specific cytostatic signals target Rb and p21, leading to G1 cell cycle arrest (hSSTR5>hSSTR2 >hSSTR4∼hSSTR1). These effects are pertussis toxin- and G protein dependent and are PTP mediated. The marked decrease in the ability of C tail mutants of hSSTR5 to induce Rb and G1 cell cycle arrest suggests that the C-terminal domain of hSSTR5 is involved in cytostatic antiproliferative signaling.
MATERIALS AND METHODS
Materials
The SST analog SMS 201–995 (octreotide, OCT) was obtained from Sandoz Pharmaceutical Co. (Basel, Switzerland). (PI) was purchased from Sigma Chemical Co. (St. Louis, MO). Rabbit polyclonal antibodies against p21 (C-19) and Rb (C-15) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). FITC-conjugated goat antimouse and antirabbit IgG antibodies were supplied by Zymed Laboratories (San Francisco, CA). All other reagents were obtained from local commercial sources and were of analytical quality.
CHO-K1 Cells Stably Expressing hSSTR1–5 and Mutant hSSTR5
Genomic fragments of hSSTR 2, 3, and 5 or cDNA clones for hSSTR 1 and 2A containing the entire coding sequences were subcloned into the polylinker region of the mammalian expression vector pRc/CMV (Invitrogen, San Diego, CA). Mutant hSSTR5 receptors with progressive truncation of the C tail (Δ347, Δ338, Δ328, and Δ318) were created by introducing stop codons at positions 347, 338, 328, and 318 of a cassette cDNA construct using the PCR overlap extension technique (25); the mutant cDNAs were cloned into the mammalian expression vector PTEJ8 (25). Wild-type hSSTRs and the mutant hSSTR5 receptors were stably transfected in CHO-K1 cells maintained under G418 selection (11, 44, 45). The binding characteristics of the different hSSTRs and the hSSTR5 C tail deletion mutants are compared in Tables 1 and 2. These values were estimated from saturation binding analysis using[ 125I-LTT]SST-28 as the radioligand as previously described (25, 44, 45). Cells were grown in T75 flasks in Hams F-12 medium containing 5% FCS (Life Technologies, Grand Island, NY) and 400 U/ml G-418 and cultured for 3–5 days at 37 C in a humidified atmosphere with 5% CO2. When the cells had reached 60–70% confluency, medium was replaced with fresh medium containing 100 nm of either OCT (hSSTRs 2, 3, and 5) or d-Trp8 SST-14 (hSSTR 1 and 4 subtypes to which OCT does not bind) (11, 44). To investigate the G protein dependency of hSSTR5-mediated cytostatic signaling, cells were preincubated for 18 h with pertussis toxin. To determine whether such action was PTP mediated, the effect of OCT was compared in the absence and presence of the tyrosine phosphatase inhibitor Na orthovanadate. The pertussis toxin and Na orthovanadate were used at optimal concentrations of 100 ng/ml and 10 mg/ml, as determined in earlier studies (11, 46). After 24 h incubation, the cells were washed in PBS, scraped, and fixed sequentially in 1% paraformaldehyde and 70% ethanol. Cellular DNA was labeled with the intercalating dye PI (50 mg/ml) in PBS and incubated at 37 C for 5 min in the presence of RNAse A (50 mg/ml). Rb and p21 were immunolabeled with their respective antibodies, followed by counterstaining with FITC-conjugated secondary antibodies as previously described (11).
Binding Characteristics of hSSTR Subtypes Expressed in CHO-K1 Cells
| Receptor | Kd (nm) | Bmax (fmol/mg) |
|---|---|---|
| hSSTR1 | 1.23 ± 0.40 | 174 ± 40 |
| hSSTR2 | 1.11 ± 0.32 | 260 ± 61 |
| hSSTR3 | 1.88 ± 0.27 | 294 ± 44 |
| hSSTR4 | 0.93 ± 0.09 | 256 ± 37 |
| hSSTR5 | 0.31 ± 0.01 | 162 ± 42 |
| Receptor | Kd (nm) | Bmax (fmol/mg) |
|---|---|---|
| hSSTR1 | 1.23 ± 0.40 | 174 ± 40 |
| hSSTR2 | 1.11 ± 0.32 | 260 ± 61 |
| hSSTR3 | 1.88 ± 0.27 | 294 ± 44 |
| hSSTR4 | 0.93 ± 0.09 | 256 ± 37 |
| hSSTR5 | 0.31 ± 0.01 | 162 ± 42 |
Receptor binding capacity was quantitated using[ 125I-LTT]SST-28 as the radioligand in saturation binding assays (mean ± se, n = 3).
Binding Characteristics of hSSTR Subtypes Expressed in CHO-K1 Cells
| Receptor | Kd (nm) | Bmax (fmol/mg) |
|---|---|---|
| hSSTR1 | 1.23 ± 0.40 | 174 ± 40 |
| hSSTR2 | 1.11 ± 0.32 | 260 ± 61 |
| hSSTR3 | 1.88 ± 0.27 | 294 ± 44 |
| hSSTR4 | 0.93 ± 0.09 | 256 ± 37 |
| hSSTR5 | 0.31 ± 0.01 | 162 ± 42 |
| Receptor | Kd (nm) | Bmax (fmol/mg) |
|---|---|---|
| hSSTR1 | 1.23 ± 0.40 | 174 ± 40 |
| hSSTR2 | 1.11 ± 0.32 | 260 ± 61 |
| hSSTR3 | 1.88 ± 0.27 | 294 ± 44 |
| hSSTR4 | 0.93 ± 0.09 | 256 ± 37 |
| hSSTR5 | 0.31 ± 0.01 | 162 ± 42 |
Receptor binding capacity was quantitated using[ 125I-LTT]SST-28 as the radioligand in saturation binding assays (mean ± se, n = 3).
Binding Characteristics of Wild-Type and Mutant hSSTR5 Receptor
| Kd (nm) | Bmax (fmol/mg) | |
|---|---|---|
| wt hSSTR5 | 0.31 ± 0.01 | 162 ± 42 |
| Δ 318 hSSTR5 | 0.89 ± 0.07 | 262 ± 71 |
| Δ 328 hSSTR5 | 0.47 ± 0.16 | 298 ± 99 |
| Δ 338 hSSTR5 | 0.41 ± 0.18 | 247 ± 40 |
| Δ 347 hSSTR5 | 0.21 ± 0.07 | 352 ± 96 |
| Kd (nm) | Bmax (fmol/mg) | |
|---|---|---|
| wt hSSTR5 | 0.31 ± 0.01 | 162 ± 42 |
| Δ 318 hSSTR5 | 0.89 ± 0.07 | 262 ± 71 |
| Δ 328 hSSTR5 | 0.47 ± 0.16 | 298 ± 99 |
| Δ 338 hSSTR5 | 0.41 ± 0.18 | 247 ± 40 |
| Δ 347 hSSTR5 | 0.21 ± 0.07 | 352 ± 96 |
Receptor binding capacity was quantitated using[ 125I-LTT]SST-28 as the radioligand in saturation binding assays (mean ± se, n = 3) (25).
Binding Characteristics of Wild-Type and Mutant hSSTR5 Receptor
| Kd (nm) | Bmax (fmol/mg) | |
|---|---|---|
| wt hSSTR5 | 0.31 ± 0.01 | 162 ± 42 |
| Δ 318 hSSTR5 | 0.89 ± 0.07 | 262 ± 71 |
| Δ 328 hSSTR5 | 0.47 ± 0.16 | 298 ± 99 |
| Δ 338 hSSTR5 | 0.41 ± 0.18 | 247 ± 40 |
| Δ 347 hSSTR5 | 0.21 ± 0.07 | 352 ± 96 |
| Kd (nm) | Bmax (fmol/mg) | |
|---|---|---|
| wt hSSTR5 | 0.31 ± 0.01 | 162 ± 42 |
| Δ 318 hSSTR5 | 0.89 ± 0.07 | 262 ± 71 |
| Δ 328 hSSTR5 | 0.47 ± 0.16 | 298 ± 99 |
| Δ 338 hSSTR5 | 0.41 ± 0.18 | 247 ± 40 |
| Δ 347 hSSTR5 | 0.21 ± 0.07 | 352 ± 96 |
Receptor binding capacity was quantitated using[ 125I-LTT]SST-28 as the radioligand in saturation binding assays (mean ± se, n = 3) (25).
Flow Cytometry
Flow cytometry was carried out in an EPICS 750 series flow cytometer (Coulter Electronics, Hialeah, FL). Fluorescence was excited by a 5-watt argon laser generating light at 351–363 nm. PI emission was detected through a 610-nm long pass filter, and FITC fluorescence was detected with a 560-nm short pass dichroic filter. At least 10,000 gated events were recorded for each sample, and the data were analyzed by Winlist software (Verity Software House).
Analysis of Nuclear Morphology
Aliquots of cells stained with PI for analysis by flow cytometry were cytospun onto microscope slides, mounted using Immunomount (Shandon, Pittsburgh, PA), viewed, and photographed through a Reichert Polyvar 2 fluorescence microscope (original magnification× 400).
Western Blot Analysis
Cells were lysed in Tris-HCl buffer (100 mm, pH 7.2) containing 300 mm NaCl, 2% Nonidet P-40, 20% glycerol, 2 mm ZnCl2, 10 mg/ml pepstatin, and 0.2 mm pefabloc (Boehringer Mannheim, Canada). Protein measurement was performed using the Bio-Rad protein assay kit (Bio-Rad Laboratories, Hercules, CA). Aliquots (30 μg) were electrophoresed in 10% SDS-polyacrylamide gel in running buffer (50 mm Tris-HCl, 60 mm boric acid, 1 mm EDTA, 0.1% SDS) and transferred onto Protran plus membranes electrophoretically in a buffer containing 25 mm Tris, 192 mm glycine, and 15% methanol. Blots were probed with anti-RB antibody (Pharmingen, San Diego, CA) and visualized with alkaline phosphatase conjugate detection kit (Bio-Rad). Molecular size was determined using 10 kDa protein ladder (Life Technologies) and staining with Ponceau S (46).
Measurement of PTP Activity
Phosphatase activity in whole-cell extracts or membrane and cytosolic fractions was determined using pNPP as the substrate as described previously (47).
Acknowledgments
We thank Ms. J. Cai for technical assistance with cell culture, Dr. N. Hukovic for binding studies with hSSTR5 mutants, and Ms. S. Schiller and Dr. Halwani for assistance with flow cytometry.
This work was supported by grants from the Medical Research Council of Canada (MT 12603 and MT 10411) and the US Department of Defense. K.S. is a recipient of a studentship award of the Fonds de la Recherche en Sante du Quebec.
