Calcitonin Receptor-Mediated Growth Suppression of HEK-293 Cells Is Accompanied by Induction of p21WAF1/CIP1 and G2/M Arrest

Andreas Evdokiou, Liza-Jane Raggatt, Gerald J. Atkins and David M. Findlay

Department of Orthopaedics and Trauma University of Adelaide The Royal Adelaide Hospital and the Hanson Centre for Cancer Research at the Institute for Veterinary and Medical Science Adelaide 5000, South Australia, Australia


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
We investigated the mechanisms by which calcitonin (CT) suppresses cellular proliferation, using HEK-293 cells stably transfected with either the rat C1a CT receptor (CTR) or the insert-negative form of the human CTR. CT treatment of clonal cell lines expressing either receptor type, but not untransfected HEK-293 cells, strongly suppressed cell growth in a concentration-dependent manner. The reduction in cell growth with CT treatment could not be attributed to cellular necrosis or apoptotic cell death, the latter assessed by both DNA fragmentation analysis and caspase 3 (CPP-32) assay. Growth inhibition was associated with an accumulation of cells in the G2 phase of the cell cycle. CT treatment of the human and rat CTR-expressing cell lines resulted in a rapid and sustained induction of mRNA encoding the cyclin-dependent kinase inhibitor, p21WAF1/CIP1, increased levels of which were maintained at least 48 h after initiation of treatment. Western blot analysis showed a rapid corresponding increase in p21WAF1/CIP1 protein, whereas protein levels of another member of the cyclin-dependent kinase inhibitor family, p27kip1, were unchanged. In parallel with the induction of p21, CT treatment reduced levels of p53 mRNA and protein. CT treatment resulted in a specific cell cycle block in G2, which was associated with inhibition of Cdc2/cyclin B kinase activity as measured by histone H1 phosphorylation. There was no evidence for p21 association with this complex despite the inhibition of Cdc2 activity. Evidence that p21 induction was causative of cell growth suppression was obtained from p21 antisense oligonucleotide experiments. Treatment with a p21 antisense oligonucleotide blocked induction of p21 expression and significantly reduced the CT-mediated growth inhibition. These observations suggest that p21 is required for the G2 arrest in response to CT, but argue against a direct role of p21 in the inhibition of Cdc2 activity. These studies suggest a novel regulation of cell cycle progression by CT and will provide a basis for detailed examination of the molecular mechanisms involved.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Calcitonin (CT) is a 32-amino acid peptide hormone of thyroidal origin, whose main recognized physiological role is the inhibition of bone resorption by acting directly on osteoclasts (1). However, both CT and its receptors (CTR) have also been identified in a large number of other cell types and tissue sites (2), suggesting roles for the CT/CTR system distinct from those involving calcium homeostasis. The CTR belongs to a subclass of the large seven-transmembrane domain (7TMD) G protein-coupled receptor (GPCR) family, which includes the receptors for PTH/PTH-related peptide (PTHrP), glucagon, vasoactive intestinal peptide, pituitary adenylate cyclase activating peptide, and other peptide agonists (3). There is now growing evidence for an important role for GPCRs in influencing cell growth and differentiation, which is of particular relevance to development, tissue remodeling and repair, and oncogenesis (4). Gains of function mutations in receptors of this class have been shown to lead to inappropriate cell growth. For example, Jansen’s metaphyseal chondrodysplasia was recently shown to be due to constitutive activity of the PTH/PTH-related peptide receptor, resulting in impairment of growth and differentiation of chondrocytes in developing long bone growth plates (5). In addition, inappropriate activity of GPCRs has been implicated in carcinogenesis, with mutant TSH receptors, for example, serving as definitive examples of oncoproteins for this receptor class (6).

Multiple isoforms of the rat and human CTRs have been described, which result from alternative splicing of the primary mRNA. In the case of the rat CTR, two forms, termed C1a and C1b, differ structurally in that C1b contains a 37-amino acid sequence in the second extracellular domain, which is not present in the C1a form and which results in different ligand binding properties (7). The human CTR (hCTR) is primarily expressed as two functionally different isoforms, comprising an insert-negative form and a form that contains 16 additional amino acids inserted in the first intracellular loop (8, 9). Unlike the insert-negative form, the insert-positive form does not signal via phospholipase C/Ca2+-mediated pathways, although it retains some signaling capacity via coupling to adenylate cyclase (8, 9). CTRs are expressed in a number of human cancer cell lines, including those of lung (10), prostate (11), and breast cancer origin (12, 13). In addition, we have recently demonstrated the presence of mRNA encoding the hCTR in all cases in a series of primary human breast tumors (14). These results were of interest because of a longstanding observation by Ng and co-workers (15), showing that CT treatment of T47D and MCF7 human breast cancer cells in culture potently reduced the proliferation of these cells. Further evidence for a role for CT in cell growth modulation was the more recent finding of a mitogenic action of CT in human prostate cancer cells (11).

The mechanisms by which 7TMD receptors influence cell growth are not well understood and are likely to be receptor- and cell type-specific. Work to date has largely focused on the involvement of immediate postreceptor signaling events (16, 17) and the modulation of growth-related early response genes (18, 19). However, since proliferation of eukaryotic cells depends on progression through the cell cycle, proteins that exert control at cell cycle checkpoints are likely to be important, as has been found in many other cellular contexts. Increased levels of these proteins, which include the cyclin-dependent kinase inhibitors (CKIs), are in general associated with growth arrest and/or differentiation, although the particular protein involved appears to be cell type dependent. Indeed, in PTH-induced differentiation of osteoblasts, which was accompanied by an inhibition of cell proliferation, a specific increase in the CKI, p27Kip1, was associated with an accumulation of the number of cells in G1 phase (20). Likewise, suppression of cell growth is frequently found to be mediated by induction of the CKI, p21WAF1/CIP1, by p53-dependent (21) or p53-independent means (22). The aim of the present work was to elucidate the mechanisms by which CT affects cell proliferation, using stably transfected HEK-293 cell lines expressing single isoforms of the human or rat CTR. The results suggest that CT inhibits progression from the G2 phase of the cell cycle by mechanisms involving the rapid and sustained induction of p21WAF1/CIP1 and inhibition of Cdc2 kinase activity.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cell Proliferation-Dose Response and Time Course
Our recent finding that the CTR gene is frequently expressed in human primary breast cancer (14), and the longstanding demonstration of the ability of CT to inhibit the growth of the CTR-bearing breast cancer cell lines (15), prompted this study of the mechanisms of CT-induced growth suppression. However, cloning of the CTR revealed that the human and rat receptors exist in at least two functionally different forms, arising by alternative splicing of the primary mRNA transcript (8, 9), and we found that mRNA corresponding to both receptor forms is expressed in T47D and MCF-7 cells. To avoid this complexity, the experiments described here were performed in HEK-293 cells transfected with single isoforms of the rat or hCTR. HEK-293 cells were chosen because the functional characteristics of the CTR have been carefully explored in this cell type (23, 24).

HEK-293 cells stably transfected with the C1a isoform of the rat CTR (clone D11) were incubated with salmon CT (sCT) under conditions of optimal mitogenic stimulus (10% FBS). A single addition of sCT profoundly inhibited the proliferation of these cells in a concentration- (Fig. 1AGo) and time- (Fig. 2Go) dependent manner. sCT did not influence the growth of untransfected HEK-293 (Figs. 1AGo and 2Go). sCT also reduced the growth rate of HEK-293 cells transfected with the insert-negative isoform of the hCTR (HR12) (Figs. 1BGo and 2Go), suggesting that the ability of the CTR to modulate cell growth is not species dependent. Similar CT-induced growth suppression was also observed in a number of independent HEK-293 clones expressing the human or rat CTR (not shown). The growth suppression observed was dependent on receptor activation, since the sCT[8–32] analog, which binds to rat and hCTRs but does not elicit intracellular signaling (24), had no effect on the rate of cell growth (Table 1Go). Interestingly, human CT (hCT), even at higher concentrations, had only a minimal effect on the growth of D11 cells and a small, though significant, effect on HR12 cells (Table 1Go), despite being a potent activator of adenylate cyclase at both the human and rat CTR (24). We speculate that the difference between sCT and hCT, in terms of growth inhibition, may relate to the different kinetics of receptor interaction of the two ligands. Whereas sCT binds in a poorly reversible manner to the CTR (10) and activates intracellular signaling persistently, receptor-bound hCT is readily dissociable from CTRs (25).



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Figure 1. The Effect of CT Concentration on Cell Proliferation

A, HEK-293 cells transfected with the rat C1a CTR (D11) (•), or untransfected HEK-293 cells ({blacksquare}), were plated at 5 x 104 cells per well in 24-well plates. After 24 h, sCT was added once at the indicated concentrations, and cells were incubated for a further 72 h. B, HEK-293 cells transfected with the hCTR (HR12) ({blacktriangleup}) were treated with sCT as described in panel A. Cells were harvested and counted on a hemocytometer. Each data point indicates the mean ± SEM of triplicate determinations. These results are representative of four experiments.

 


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Figure 2. The Effect of CT on Cell Proliferation

HEK-293 ({blacksquare}), D11 (•), and HR12 ({blacktriangleup}) cells were seeded at 2 x 104 cells per well in 24-well plates. Twenty four hours after seeding cells remained untreated (open symbols) or were treated with one addition of 10 nM sCT (closed symbols). Cells were harvested and counted on a hemocytometer 24, 48, 72, and 96 h after sCT addition. Data points represent the mean ± SEM of triplicate determinations, and the results are representative of three experiments.

 

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Table 1. Comparison of sCT, hCT, and [8–32]sCT on Cell Proliferation

 
Apoptosis Analysis
Given the potent inhibition of cell proliferation produced by CT treatment of CTR-transfected HEK-293 cells, we sought to determine whether this might be mediated by increased cell death. Trypan blue staining of CT- treated cells showed no difference in cell viability between CT-treated and control cells. When the nuclei of CT-treated cells were stained with 4',6-diamidino-2-phenylindole (DAPI) and examined by confocal fluorescence microscopy, we found no evidence of morphological changes typical of apoptosis, i.e. condensed and fragmented nuclei. Consistent with these results, there was also no indication of CT-induced apoptosis, as determined by caspase 3 (CPP-32) assay (data not shown). When DNA was isolated and subjected to agarose gel electrophoresis, no internucleosomal DNA fragmentation could be detected, in either CT-treated or control cells (Fig. 3Go). In contrast, DNA isolated from D11 cells expressing the rat CTR treated with sCT under low serum conditions showed the characteristic DNA laddering indicative of apoptosis (Fig. 3Go). Similar results were obtained with cells expressing the hCTR (data not shown). We conclude that CT does not influence cell viability in HEK-293 cells under serum-replete conditions but may increase the susceptibility of these cells to apoptotic death by a variety of other stress signals including growth factor deprivation.



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Figure 3. Detection of DNA Fragmentation after Treatment with CT

Parental HEK-293 and D11 cells were untreated or treated once with 10 nM sCT, hCT, or sCT[8–32]. DNA was isolated and subjected to agarose gel electrophoresis. No internucleosomal DNA fragmentation was observed when cells were grown in the presence of 10% serum. However, a low molecular-size DNA ladder, which is characteristic of cells undergoing apoptosis, was observed in D11 cells grown in the presence of sCT in 0.5% serum.

 
Cell Cycle Analysis
The lack of effect of CT on cell viability suggested that CT was acting to inhibit passage of cells through the cell cycle. Analysis by fluorescence-activated cell scanning (FACS) was performed to determine the effect of CT on the distribution of cells in each phase of the cell cycle. Treatment of D11 or HR12 cells with 10 nM sCT for 72 h elicited a prominent accumulation of cells in the G2/M phase of the cell cycle and a concomitant decreased proportion of cells in G1, compared with untreated cells (Fig. 4Go). Untransfected cells were not influenced by CT treatment. These observations are consistent with the notion that CT causes G2/M arrest. The time course of these changes is shown in Fig. 5Go, which indicates that G2/M arrest was seen as early as 24 h after a single addition of CT and that maximal effects on G2/M were seen 72 h after CT addition.



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Figure 4. FACS Analysis of CT-Treated Cells

HEK-293, D11, and HR12 cells were untreated or treated with one addition of sCT (10 nM). Cells were harvested 72 h later, fixed, stained, and analyzed for DNA content, as described in Materials and Methods. Shown is the distribution and percentage of cells in the G1, S, and G2 phase of the cell cycle, respectively.

 


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Figure 5. The Effect of CT on Cell Cycle Progression

HEK-293 ({square}, {blacksquare}) and D11 ({circ}, •) cells were plated in six-well plates at 8 x 104 cells per well. Forty eight hours after plating, cells were left untreated (open symbols) or were treated once with 10 nM sCT (closed symbols). Cells were harvested and fixed at the indicated times after sCT addition and stained and analyzed for DNA content, as described in Materials and Methods. Each data point represents the percentage of cells in the G1, S, or G2 phase of the cell cycle, respectively. The results are representative of two independent experiments.

 
CT Elevates Both p21 mRNA and Protein in CTR-Transfected Cells
To examine the mechanism(s) underlying CTR-mediated growth suppression, the effects of CT on the expression of two members of the Cip/Kip family of cyclin-dependent kinase (cdk) inhibitors, p21Waf1/Cip1 and p27Kip1, were examined by Northern blot and Western blot analysis. Treatment of D11 cells with sCT resulted in a marked increase (~3 fold) in the levels of p21 mRNA by 4 h of treatment. The levels of p21 mRNA increased with increasing cell density in untransfected HEK-293 cells or D11 cells in the absence of CT. A single addition of sCT resulted in a further increase, which was sustained over the 48 h treatment period (Fig. 6Go, A and B). Similar effects were seen in HR12 cells, although with slightly different kinetics, so that p21 mRNA levels did not peak until 24 h after treatment. There was no effect of CT on p21 mRNA levels in untransfected HEK-293 cells. In agreement with its failure to affect growth of CTR-expressing HEK cells, sCT[8–32] did not influence p21 mRNA levels (data not shown).



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Figure 6. Expression of p21 and p53 mRNA after Treatment with CT

Parental HEK-293 cells and cells stably transfected with either C1a isoform of the rat CTR (clone D11) or the insert negative isoform of the hCTR (clone HR12) were cultured for the times indicated in the absence or presence of 10 nM sCT, added once. Total RNA was extracted and Northern blot analysis performed. Panel A, 10 µg of total RNA were electrophoresed through a 1% agarose formaldehyde gel, transferred to nylon membrane, and hybridized with cDNA probes for p21 and p53 as indicated. Blots were rehybridized with a cDNA probe specific for GAPDH to indicate RNA loading. Results were analyzed by densitometry and expressed as a ratio of p21 mRNA/GAPDH mRNA (panel B) and p53 mRNA/GAPDH mRNA (panel C).

 
To determine whether the increase of p21 mRNA was reflected in the level of p21 protein, Western blot analysis was performed using a p21 monoclonal antibody. A corresponding large increase (~20 fold over untreated cells) in the levels of p21 protein were detected in the D11 CTR transfectants within 4 h of CT treatment (Fig. 7AGo). By 24 h of CT treatment, the levels of p21 protein had declined but were consistently higher than in untreated cells at this time. There was no effect of CT in untransfected cells. In contrast to the observations with p21, CT treatment had no appreciable effect on p27 protein levels, as determined by Western blot analysis (Fig. 7BGo).



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Figure 7. Western Blot Analysis of p21 and p27 Protein after Treatment with CT

Parental HEK-293 and D11 cells were untreated or treated once with 10 nM sCT for the times indicated. Total cell lysates were prepared, and equal amounts of total cell protein (50 µg) were separated by SDS-PAGE, transferred to PVDF membrane, and immunoblotted with mouse monoclonal antibodies against p21 protein (panel A), p27 protein (panel B), and p53 protein (panel C). In each case, results were analyzed by densitometry and expressed as bar charts.

 
Since the involvement of p21 in the control of cell growth is dependent on p53 in some cell systems, steady state levels of p53 mRNA were measured concomitantly with p21 mRNA. CT treatment resulted in a consistent decrease in p53 mRNA levels in both D11 and HR12 cells (Fig. 6Go, A and C). No change was seen in untransfected HEK-293 cells. Similarly, the levels of p53 protein declined rapidly within 2 h after treatment of HR12 cells with CT (Fig. 7CGo).

The Effect of CT on Mitotic Index and p34cdc2 Kinase Activity
To investigate whether the CT-induced G2/M accumulation was due to a specific block in G2 or in M phase, the mitotic index of CT-treated cells was evaluated 24 h after CT treatment. The number of mitotic cells was 10-fold lower in CT-treated cells (3.6 ± 0.4) compared with controls (32.7 ± 5.1), indicative of a specific block in G2 and failure of cells to enter mitosis. Because cyclin B1 protein levels reportedly increase as cells approach M phase (26), we determined the level of cyclin B1 in HR12 cells cultured in the presence or absence of sCT. As shown in Fig. 8AGo, culture of HR12 cells in the presence of sCT resulted in a time-dependent increase in the level of cyclin B1 protein, with a peak at 48 h after treatment, coincident with the accumulation of cells in late G2 (Fig. 5Go).



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Figure 8. Effect of CT on Cyclin B1 Protein Levels and on Cdc2 Protein Kinase Activity in HR12 Cells

HR12 cells were untreated or treated with sCT for different times, and total cell extracts (50 µg) were assayed for cyclin B1 by Western blotting (panel A). Aliquots of the same extracts were immunoprecipitated with a p21 polyclonal antibody, and immune complexes were resolved by electrophoresis and assayed for p21 (panel B). For Cdc2 kinase activity, cells were treated with CT for the indicated times or treated with the drug nocodazole for 24 h. Immune complexes were resolved by electrophoresis and immunoblotted with Cdc2 (panel C, upper panel). The Cdc2 immunoprecipitated complexes were assayed further for their ability to phosphorylate histone H1 in vitro (panel C, lower panel). For cyclin B1-associated kinase activity, extracts were immunoprecipitated with a cyclin B1 polyclonal antibody, and kinase activity was assessed as previously described (panel C, lower panel). Phosphorylation of Cdc2 Tyr15 in total cell lysates (50 µg) treated with CT or nocodazole was detected by Western blotting using a phospho-Cdc2 (Tyr15)-specific antibody (panel D).

 
A recognized explanation for G2 arrest is the inhibition of Cdc2 kinase, an enzyme essential for the onset of mitosis in mammalian cells. Inhibition of Cdc2 is a function of its phosphorylation status (26) and association of p21 with the Cdc2/cyclin B complex (27). To define further the kinetics of p21 up-regulation after CT treatment, Western blot analyses of p21 immunoprecipitates isolated from HR12 cells were assayed for p21 protein. Figure 8BGo shows that p21 was barely detectable in immunoprecipitates of untreated cells but increased significantly within 2 h after CT treatment, peaked at 4 h, and remained markedly elevated in CT-treated cells when compared with untreated cells. The induction of p21 before the onset of G2 made it possible that p21 had associated with Cdc2 and prevented its activation. However, when Cdc2 or cyclin B1 immunoprecipitates isolated from CT-treated HR12 cells were assayed for the presence of p21 protein, we were unable to detect p21 associated with Cdc2 or cyclin B1 at any time after CT treatment (data not shown). In the converse experiment, Cdc2 was undetectable in the corresponding p21 immunoprecipitates. To confirm that Cdc2 was successfully immunoprecipitated from the lysates, Cdc2-immunoprecipitated complexes were resolved by electrophoresis and assayed for Cdc2 by Western blotting (Fig. 8CGo, upper panel). Cdc2 was readily detectable in the immune complexes, and the levels were not altered after treatment with CT. To assess the kinase activity of Cdc2 at different times after addition of CT, Cdc2 and cyclin B1 immunoprecipitates from HR12 cells were analyzed for their ability to phosphorylate histone H1. Cdc2- and and cyclin B1-associated kinase activity (Fig. 8CGo) increased transiently in the first 24 h after CT treatment but declined significantly thereafter and was lowest at 48 and 72 h, concomitant with accumulation of cells in G2. In contrast, when cells were specifically arrested in mitosis with the microtubule-depolymerizing drug, nocodazole, the histone H1 kinase activity associated with Cdc2 was considerably higher than that in the CT-treated cells (Fig. 8CGo). To investigate the inactivation of Cdc2 by CT on Tyr15 phosphorylation, cell extracts were immunoblotted with a phospho-Cdc2Tyr15-specific antibody, which detects Cdc2 when catalytically inactivated by phosphorylation at Tyr15. As shown in Fig. 8DGo, CT treatment resulted in a modest but reproducible increase in Cdc2Tyr 15 phosphorylation, which occurred at 48 and 72 h posttreatment, consistent with inactive Cdc2 and G2 arrest. In contrast, cells treated with nocodazole had unphosphorylated Cdc2Tyr15. These data indicate that when M phase was blocked by activation of the spindle microtubule-assembly checkpoint using nocodazole, Cdc2 complexes were held in an active state manifested by the lack of Cdc2 Tyr15 phosphorylation (Fig. 8DGo). However, when cells were arrested in G2 with CT, the kinase activity of Cdc2 complexes was low, and Cdc2 Tyr15 was maintained in the phosphorylated and inactive state. Taken together, these results clearly demonstrate that CT blocks progression into mitosis by inhibiting the kinase activity of the Cdc2/cyclin B1. Since this effect appears to be independent of p21, the exact role of p21 in the CT-mediated G2 arrest remains to be determined.

Effect of Antisense p21 on Cell Growth after CT Treatment
The kinetics of p21 mRNA and protein up-regulation, peaking early at 2 h after CT treatment, suggest that p21 participates in the observed G2 arrest. To investigate whether p21 induction by CT is a cause or a result of G2 arrest, we assessed CT-induced growth inhibition in the presence of a p21 sense or antisense oligonucleotide. As expected, incubation of hCTR-expressing cells (HR12) with the sense oligonucleotide did not influence the increase in p21 mRNA after CT treatment. However, incubation with the p21 antisense oligonucleotide abolished the ability of CT to increase p21 mRNA (Fig. 9AGo) and protein (Fig. 9BGo). The effect of CT on cell growth in the presence of the p21AS oligonucleotide was determined in four independent experiments carried out in triplicate. As shown in Fig. 9CGo, the p21AS oligonucleotide significantly prevented the CT-mediated growth inhibition, while the p21S oligonucleotide did not. There was no significant difference between cell numbers in control, sense-, or antisense oligonucleotide-treated cells, in the absence of CT, ruling out nonspecific toxicity of the oligonucleotides. These results suggest a causative role of p21 in CT-mediated growth inhibition.



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Figure 9. Effect of Antisense p21 on the Growth of HR12 Cells after CT Treatment

HR12 cells were untreated (-) or treated (+) with CT in the presence or absence of either the sense or antisense p21 oligonucleotide. Northern blot analysis was performed to assess the levels of p21 mRNA at 24 and 48 h after treatment with CT (panel A). Cell extracts were collected 48 h after CT treatment, and p21 protein levels were assessed by Western blotting using a p21-specific antibody (panel B). HR12 cells were trypsinized and counted 48 h post-CT treatment (panel C). These results are representative of four independent transfection experiments.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
CT is best understood in its role as a potent negative regulator of osteoclastic bone resorption (2). However, the more recent discovery of CT production (2, 28), and expression of its receptor (2, 8), in extraskeletal sites implies that CT may have actions unrelated to calcium metabolism and protection of the skeleton. Relevant to the present report is the longstanding finding of CT-induced growth suppression in T47D and MCF7 human breast cancer cells (15). Together with evidence for a mitogenic action of CT in a prostate cancer cell line (11), these data draw attention to the specific ability of CT to modulate cell growth and the more general involvement of the 7TMD class of receptor in cell proliferation (4). The present study shows that sCT treatment of HEK-293 cells stably transfected with the rat C1a CTR, or the insert-negative form of the hCTR, potently decreased cellular proliferation. Treatment with hCT, on the other hand, had a minimal effect on the growth of D11 cells expressing the rat CTR and a significant, but much lesser, effect than sCT on the growth of HR12 cells expressing the hCTR. There are several possible implications of this finding. First, hCT may be antiproliferative only at the hCTR, as seen also in T47D and MCF-7 breast cancer cells (15). In these experiments, hCT was added repeatedly and inhibition of cell growth occurred over a longer time course than the present experiment. Second, the teleost or fish-like CTs may be more potent than hCT in terms of growth inhibition. There is good evidence for the presence of fish-like CTs in several mammalian tissues, including brain and pituitary (2), where their physiology is not yet understood. Moreover, our results suggest the possibility of using sCT therapeutically as an antiproliferative agent in human cancer, given our finding of the frequent expression of the CTR in primary breast tumors (14).

It may be that molecules in the CT family are involved physiologically in cellular decisions regarding growth and differentiation, as has been reported for several ligands for other members of the receptor subclass to which the CTRs belong. For example, pituitary adenylate cyclase activating peptide (PACAP) (16), vasoactive intestinal peptide (17), and PTH (18) have also been shown to modulate cell growth, in addition to their reported acute effects on differentiated cells. In the case of PTH, there is in vitro (18, 29) and in vivo (30) evidence for an important role in cell growth and differentiation. PTH receptor deficiency (31) or constitutively active receptor (5) lead to altered chondrocyte growth and differentiation, manifested as deranged growth plates in long bones. Studies to date, examining the mechanism by which this class of receptor alters cellular growth, have largely focused on signaling events proximal to receptor activation. The signaling pathways that regulate growth are complex and apparently receptor- and cell-type specific (32). It was shown previously that CT inhibition of T47D cell growth occurred in parallel with a selective and sustained activation of the type II isoform of protein kinase A (15). However, a more recent report of CT signaling in HEK-293 cells showed Gq-mediated stimulation of Erk1/2 activity (23). These results are significant in the present context because activation of the mitogen-activated protein kinase pathway is known to correlate with inhibition of cell proliferation (33). CT has been shown to regulate the early response genes, c-fos and c-jun, in several cell types (18, 19); however, the nuclear actions of CT on cell growth have not previously been explored.

Inhibition of growth by CT in transfected HEK-293 cells was accompanied by a rapid and prolonged induction of the cyclin-dependent kinase inhibitor, p21Waf1/Cip1. P21Waf1/Cip1 has largely been studied with respect to inhibition of cyclin-cdk complexes involved in cell cycle arrest in the G1 phase of the cell cycle, viz, cyclin D-cdk and cyclin E-cdk (e.g. Refs. 22, 34). However, early experiments to assay the activity of p21Waf1/Cip1 suggested that it was potentially a universal inhibitor of cyclin kinases (35), and p21 mRNA in human fibroblasts shows bimodal periodicity, with peaks in both G1 and G2/M (36). Expression in the G2/M phase suggests a role for p21Waf1/Cip1 in the control of the cell cycle during this particular phase. In the experiments reported here, we found that CT treatment of CTR-transfected HEK-293 cells resulted in a prominent and prolonged accumulation of cells in G2/M phase of the cell cycle, with no detectable G1 arrest. Despite the significant accumulation of cells with a G2/M DNA content, the percentage of mitotic cells was 10-fold lower in CT-treated cells as compared with controls, suggesting a specific inhibition of cells in G2 and not in M. The results of the p21 antisense oligonucleotide experiments suggest that p21Waf1/Cip1 is causally involved in the CT-mediated growth suppression in HEK-293 cells. In addition, the early elevation of p21Waf1/Cip1, preceding inhibition of cell growth and cell cycle progression, and its sustained high levels in CT-treated cells, is consistent with the activity of this protein in many other cellular systems in which growth inhibition is observed. However, the role of p21 at the G2 checkpoint remains to be clarified. We obtained no evidence for the presence of p21 protein in the Cdc2/cyclin B1 complex despite inhibition of Cdc2 kinase activity, arguing against a direct role for p21 in inhibiting Cdc2 kinase. Thus the significance of a sustained induction of p21 and the molecular mechanisms underlying the observed cell cycle arrest in G2 after CT treatment remain to be clarified.

The association of Cdc2 kinase with its cyclin partners has been shown to play an important role during G2 to M progression in mammalian cells (37). The activity of Cdc2 is regulated positively by phosphorylation of threonine 161 (38) and negatively by phosphorylation on Tyr-15 and Thr-14. Inhibitory phosphorylation of Cdc2 on residues Tyr-15 and Thr-14 must be removed by the action of the dual specificity phosphatase Cdc25 for the Cdc2/cyclin B complex to become active as the driving force for mitosis (26). Here, we have shown that CT induces a specific block in G2 phase of the cell cycle by a mechanism involving the inhibition of Cdc2 kinase activity. Interestingly, the activity of Cdc2 increased transiently in the first 24 h after CT treatment but declined significantly thereafter and was lowest at 48 and 72 h, concomitant with cells accumulating in G2. This transient increase in Cdc2 kinase activity was reproducibly observed and its significance will require further investigation.

The promoter for the p21Waf1/Cip1 gene contains multiple p53-response elements (39) and induction of p21Waf1/Cip1 has been shown to occur under p53-dependent conditions (40, 41). However, induction in other situations appears not to require p53 (36). In the CTR transfectants, CT induction of p21Waf1/Cip1 was unlikely to have been p53-mediated since p53 mRNA levels were regulated inversely to p21Waf1/Cip1 mRNA levels. The explanation for this, and the possible involvement of reduced p53 levels in CT-induced growth suppression, remains to be investigated. The reduced expression of p53 correlated with the arrest of CT-treated cells in G2, rather than G1, which is more typically the case when p21Waf1/Cip1 induction is secondary to up-regulation of p53 (reviewed in Ref. 42). In addition, up-regulation of p53 in some cell types is associated with the induction of apoptotic death (reviewed in Ref. 42), and the decrease in proliferation of CT-treated HEK-293 cells in serum-replete medium clearly did not precede apoptosis. It is interesting to speculate that the observed decrease in p53 mRNA and protein levels might represent a mechanism to protect the CT-treated cells from apoptosis after G2 arrest.

These studies suggest a novel regulation of cell cycle progression by CT and provide the basis for further detailed examination of the molecular mechanisms linking activation of the CTR to proliferation. The recent implication of CT/CTR in human cancers (11, 14) and early embryonic development (28, 43) serve to further stimulate this line of enquiry.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cell Culture
Human embryonic kidney (HEK-293) cells were maintained in DMEM supplemented with 10% heat-inactivated FBS, 2 mM glutamine, 100 U/ml penicillin, and 0.1 mg/ml streptomycin, as previously reported (23). Stably transfected HEK-293 cells were maintained in 200 µg/ml G418, which was removed before commencement of experiments. Cells were grown at 37 C in a humidified atmosphere with 5% CO2.

Stable Transfection of HEK-293 Cells
Two cell lines, D11 and F12, which express the rat C1a isoform of the rat CTR in HEK-293 cells, have been described previously (23). To compare the growth effects of the hCTR in the same cell type, HEK-293 cells were transfected with the insert-negative isoform of the hCTR. The hCTR cDNA was ligated into the mammalian expression vector Zem228CC (both cDNA and vector were gifts from Zymogenetics Inc, Seattle, WA). Using a modified calcium phosphate transfection method (44), cells were transfected with 10 µg plasmid DNA/flask, together with 20 µg of inert herring sperm DNA (Sigma Chemical Co., St Louis, MO), in 25-cm2 culture flasks at a cell density of approximately 50%. Twenty-four hours after transfection, neomycin selection was commenced with the addition of 400 µg/ml G418 (Life Technologies, Inc., Glen Waverley, Victoria, Australia) and maintained for 2 weeks. Neomycin-resistant colonies were picked manually, propagated in the continual presence of G418 (200 µg/ml), and screened for binding of [125I]sCT. A number of clones were obtained with different levels of receptor expression. The cell line designated HR12, which expressed 14.3 ± 3.7 x 105 receptors per cell (mean ± SEM, n = 3), and bound [125I]sCT with a Kd of 2.0 ± 0.4 nM (mean ± SEM, n = 3), was primarily used for this study. As described for the C1a rat CTR, CT treatment of HR12 cells also resulted in increased intracellular CAMP levels and intracellular Ca2+ fluxes. Several other clones expressing the hCTR yielded similar results, with respect to cell growth and induction of p21WAF1/CIP1.

Cell Growth Analysis
To determine the effect of CT concentration on cell proliferation, cells were seeded at 5 x 104 cells per well in 24-well plates and incubated for 72 h in the presence of increasing concentrations (10-11 M–10-7 M) of sCT, hCT, or the 8–32 analog of sCT (Peninsula Laboratories, Inc. Belmont, CA). To determine the effect of sCT on the rate of cell growth, cells were plated in 24-well plates at 2 x 104 cells per well in standard culture medium in the presence or absence of 1 x 10-8 M sCT for 1–4 days. Cells were harvested by trypsinization and were counted manually in triplicate using a hemocytometer.

Apoptosis Analysis
DNA Fragmentation
Cells were harvested by washing twice with PBS, after 72 h in the presence or absence of 10 nM sCT, and incubated overnight at 37 C in lysis buffer containing 10 mM Tris, pH 8.0, 5 mM EDTA, 100 mM NaCl, 1.0% SDS, and 200 µg/ml proteinase K. DNA was extracted twice with equal volumes of phenol-chloroform-isoamylalcohol (25:24:1) and then precipitated in ethanol. Samples were electrophoresed in a 1.2% agarose gel, stained with ethidium bromide, and visualized under UV light.

Caspase 3 Assay
CPP-32/caspase 3 protease activity was assayed by the cleavage of DEVD-AFC, a fluorogenic substrate based on the peptide sequence of the caspase-3 cleavage site of poly(ADP-ribose) polymerase. Cells were cultured for 48 h in the presence or absence of 10 nM sCT, washed in PBS, and resuspended in NP-40 lysis buffer. To each tube containing 8 µM of substrate in 1 ml of protease buffer (50 mM HEPES, 10% sucrose, 10 mM dithiothreitol, and 0.1% 3-[(chloramidopropyl)dimethylamino]-1-propanesulfonate, pH 7.4), was added 20–40 µl of cell lysate, and the mixture was incubated at room temperature for 45 min. Caspase activity was quantitated by measurement of yellow-green fluorescence at 505 nm (excitation at 400 nm), due to release of AFC, in a LS50 spectrofluorimeter (Perkin Elmer Corp., Norwalk, CT).

Cell Cycle Analysis
Cells were removed from culture dishes by trypsinization, collected by centrifugation, resuspended in ice-cold PBS, and then fixed in absolute methanol for at least 30 min. Cells were then washed in PBS containing 0.5% Tween 20, followed by two washes in PBS containing 2% FCS, and then cells were resuspended in PBS/2% FCS containing 40 µg/ml RNase A and incubated for 20 min at 37 C. Cells were washed in PBS/2% FCS and were finally resuspended in PBS containing propidium iodide at a final concentration of 20 µg/ml. The stained nuclei were analyzed using a flow cytometer (Epics Profile, Coulter Corp., Hialeah, FL). Cell cycle distribution was based on 2 N and 4 N DNA content.

Mitotic Index Analysis
Cells treated or not with 10 nM sCT for 24 h were trypsinized, collected by centrifugation, washed in PBS, and treated with hypotonic solution of 0.075 M KCl at 37 C for 30 min. After centrifugation, cells were fixed by the dropwise addition of freshly prepared fixative (methanol-acetic acid, 3:1), recentrifuged, and resuspended in fixative. Cells were dropped onto clean microscope slides and stained with a 1:25 dilution of Giemsa (Sigma Chemical Co.) in PBS for 30 min. Mitotic indices were calculated as the number of cells with condensed chromosomes with at least 3000 cells being examined from 10 different fields of view.

RNA Extraction and Northern Blot Analysis
For Northern blot analysis, cells were seeded at a density of 1 x 105 cells per 25-cm2 flask, allowed to attach for 2 days, and incubated for 4, 24, and 48 h in the presence or absence of 10 nM sCT. Total RNA was isolated at the indicated times using the TRIZOL Reagent (Life Technologies, Inc.) according to the manufacturer’s instructions. Total RNA (10 µg per lane) was electrophoresed in formaldehyde/1% agarose gels, transferred to Hybond N+ nylon membranes (Amersham Pharmacia Biotech, Castle Hill, New South Wales, Australia), and immobilized by UV cross-linking. Membranes were prehybridized for 3 h at 42 C in 1 M NaCl, 1% SDS, 10% dextran sulfate, 50% formamide, and 100 µg/ml of heat-denatured herring sperm DNA and hybridized with p21WAF1/CIP1 cDNA (a kind gift from Dr. Helena Richardson, The University of Adelaide, South Australia, Australia), or p53 cDNA (a kind gift from Dr. Roger Reddel, Children’s Medical Research Institute, Westmead, New South Wales, Australia) radiolabeled with [{alpha}-32P]dCTP by random priming using the Giga prime kit (Bresatec, South Australia). To allow quantitation of mRNA signals, the same filters were stripped and then reprobed with a 450-bp 32P-labeled PCR-generated DNA fragment of human glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Northern blots were analyzed using the PhosphorImager SF (Molecular Dynamics, Inc., Sunnyvale, CA).

Western Blotting
Cells were lysed in lysis buffer containing 10 mM Tris HCl, pH 7.6, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, and stored at -70 C until ready to use. Cell extracts were mixed with an equal volume of sample buffer containing 12 mM Tris-HCl, pH 6.8, 6% SDS, 10% ß- mercaptoethanol, 20% glycerol and 0.03% bromophenol blue. Protein samples were boiled for 5 min and electrophoresed under reducing conditions in 14% polyacrylamide gels. Separated proteins were electrophoretically transferred to PVDF transfer membrane (NEN Life Science Products, Boston, MA) and blocked in PBS containing 5% blocking reagent (Amersham Pharmacia Biotech, Piscataway, NJ) for 1 h at room temperature. Immunodetection was performed overnight at 4 C in PBS/blocking reagent containing 0.1% Tween 20, using mouse monoclonal antibodies to p21 (C24420), p27 (K25020) (Transduction Laboratories, Inc., Lexington, KY), p53 (clone PAB1801) (Zymed Laboratories, Inc., South San Francisco, CA) and rabbit polyclonal antibodies to cyclin B1 (H-433) and Cdc2 (C-19) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Filters were rinsed several times with PBS containing 0.1% Tween 20 and incubated with 1:5000 dilution of antimouse or antirabbit alkaline phosphatase-conjugate (Amersham Pharmacia Biotech, Arlington Heights, IL) for 1 h. Bound proteins were detected and quantitated using the Vistra ECF substrate reagent kit (Amersham Pharmacia Biotech) on a Fluorimager (Molecular Dynamics, Inc.).

Immunoprecipitation and Kinase Activity
For immunoprecipitation as for kinase activity determination, rabbit polyclonal antibodies to p34cdc2, p21, or cyclin B1 (Santa Cruz Biotechnology, Inc.) were attached to protein A-Sepharose (Pharmacia Biotech) by incubation at 4 C for 24 h. Antibody-attached Sepharose was then added to 300 µg of precleared cell lysates and incubated for a further 24 h at 4 C on a rotating platform. Immune complexes were recovered and washed three times with lysis buffer. For immunoprecipitation, immune complexes were separated on 14% SDS-polyacrylamide gels as described above, and a mouse monoclonal p21 antibody or rabbit polyclonal p34cdc2 was used for immunoblot analysis. For p34cdc2 kinase activity, immune complexes were washed three times with kinase assay buffer (50 mM Tris-HCl, pH 7.4, 10 mM MgCl2, 1 mM dithiothreitol). Phosphorylation of histone H1 was measured by incubating the beads with 40 µl of a reaction mixture containing 25 µM ATP, 2 µg histone H1 (Roche Molecular Biochemicals, Nutley, NJ) and 2.5 µCi [32P]{gamma}-ATP (3000 Ci/mmol; Bresatec) in kinase assay buffer for 30 min at 37 C. The reaction was stopped by boiling the sample in 2 x SDS sample buffer for 5 min, and samples were resolved by SDS-PAGE. Gels were analyzed using the PhosphorImager SF (Molecular Dynamics, Inc.).

Antisense Oligonucleotides
Phosphorothioate oligodeoxynucleotides (2 µg/ml) and lipofectin (Life Technologies, Inc.) (10 µg/ml) were incubated in serum free DMEM for 45 min at room temperature. The oligonucleotide-lipofectin mixture was diluted with serum-containing medium and added to the cells. After 4 h of preincubation with the oligonucleotide mixture, cells were treated with 10 nM sCT for up to 72 h. To confirm that the antisense oligonucleotide had the desired effect of blocking p21 up-regulation by sCT, p21 mRNA was monitored by Northern and Western blotting at 24 and 48 h after treatment with sCT. At the indicated times, cells were trypsinized from plates and counted. The antisense oligonucleotide was based on the p21 coding sequence p21AS, which is complementary to the region of the initiation codon (5'-TCC CCA GCC GGT TCT GAC AT-3') and the sense p21 (Sp21) as control (5'-ATG TCA GAA CCG GCT GGG GA-3'). These oligonucleotides were purchased from Life Technologies, Inc.


    ACKNOWLEDGMENTS
 
The authors are grateful for reagents and advice from Zymogenetics Inc. (Seattle, WA) and Dr. Prue Cowled and Lefta Leonardos at the Queen Elizabeth Hospital (Adelaide, South Australia, Australia). We are also grateful to Dr Matthew O’Connell at the Peter MacCallum Cancer Institute (Melbourne, Victoria, Australia) for useful discussions.


    FOOTNOTES
 
Address requests for reprints to: Associate Professor David M. Findlay, Department of Orthopaedics and Trauma, Level 4, Bice Building, Royal Adelaide Hospital, North Terrace, Adelaide 5000, South Australia, Australia.

This work was supported by The Kathleen Cunningham Foundation for Breast Cancer Research, Australia, and grants from the Adelaide Bone and Joint Research Foundation and Bristol-Myers Squibb Co./Zimmer. L.-J. Raggatt was supported by the Australian Research Council Small Grants scheme.

Received for publication December 10, 1998. Revision received June 30, 1999. Accepted for publication July 8, 1999.


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