Endocrine-Hypertension Division and Membrane Biology Program, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115
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ABSTRACT |
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Prostate cancer metastasizes
frequently to bone. Elevated extracellular calcium concentrations
([Ca2+]o) stimulate parathyroid
hormone-related protein (PTHrP) secretion from normal and malignant
cells, potentially acting via the
[Ca2+]o-sensing receptor (CaR). Because
prostate cancers produce PTHrP, if high
[Ca2+]o stimulates PTHrP secretion via the
CaR, this could initiate a mechanism whereby osteolysis caused by bony
metastases of prostate cancer promotes further bone resorption. We
investigated whether the prostate cancer cell lines LnCaP and PC-3
express the CaR and whether polycationic CaR agonists stimulate PTHrP
release. Both PC-3 and LnCaP prostate cancer cell lines expressed bona fide CaR transcripts by Northern analysis and RT-PCR and CaR protein by
immunocytochemistry and Western analysis. The polycationic CaR agonists
[Ca2+]o, neomycin, and spermine each
concentration dependently stimulated PTHrP secretion from PC-3 cells,
as measured by immunoradiometric assay, with maximal, 3.2-, 3.6-, and
4.2-fold increases, respectively. In addition, adenovirus-mediated
infection of PC-3 cells with a dominant negative CaR construct
attenuated high [Ca2+]o-evoked PTHrP
secretion, further supporting the CaR's mediatory role in this
process. Finally, pretreating PC-3 cells with transforming growth
factor (TGF)-1 augmented both basal and high
[Ca2+]o-stimulated PTHrP secretion. Thus, in
PTHrP-secreting prostate cancers metastatic to bone, the CaR could
initiate a vicious cycle, whereby PTHrP-induced bone resorption
releases [Ca2+]o and TGF-
stored within
bone, further increasing PTHrP release and osteolysis.
parathyroid hormone-related protein; ion-sensing receptor; osteolysis; prostate cancer; LnCaP cells; skeletal metastases
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INTRODUCTION |
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PROSTATE CANCER IS A COMMON CANCER and the second leading cause of cancer death in men (4). A substantial percentage of elderly men have microscopic prostate cancers, but these small lesions usually remain localized to the prostate and never come to clinical attention. Nevertheless, skeletal complications of prostate cancer are a difficult clinical problem, causing disabling pain and other complications such as fractures (10). Radiation, hormonal manipulations, and/or chemotherapy offer palliation but, unfortunately, little hope of cure for skeletal metastases of prostate cancer. Therefore, further understanding of the biology of prostate cancer metastatic to bone and the development of improved therapies of skeletal metastases and their complications are important goals of prostate cancer research.
Recent studies have shown that parathyroid hormone (PTH)-related protein (PTHrP) is a central mediator of malignancy-associated hypercalcemia and osteolysis. In addition to causing most cases of humoral hypercalcemia of malignancy, where skeletal metastases are absent, PTHrP, originally isolated from renal, lung, and breast cancers (7, 37, 39), is the biological mediator in ~70% of cases of malignant osteolysis with or without hypercalcemia, particularly that caused by common epithelial cancers [i.e., breast (16)]. Although prostate cancers metastatic to bone generally cause osteoblastic lesions, substantial increases in bone resorption also occur in this setting, as assessed by biochemical markers (10, 24, 40). Indeed, markers of bone resorption can be higher in patients with metastatic prostate cancer than in those with skeletal metastases of breast cancer (10). Prostate cancers often express more PTHrP than normal prostate epithelial cells (1, 25), suggesting that PTHrP could contribute to the increased bone resorption (10) in patients with prostate cancer metastatic to bone (1, 25, 38). PTHrP secreted by prostate cancer cells could then activate osteoclasts and potentially contribute to skeletal invasiveness, bone pain, and/or pathological fractures. Therefore, further understanding of the factors regulating the production and secretion of PTHrP by prostate cancer cells could elucidate the mechanisms underlying the excessive bone resorption associated with this tumor and potentially provide clues to novel therapeutic strategies.
The extracellular calcium ([Ca2+]o)-sensing receptor (CaR) is a G protein-coupled cell surface receptor that is a central element in [Ca2+]o homeostasis (6). In parathyroid cells, high [Ca2+]o, by activating the CaR, inhibits PTH secretion and parathyroid cellular proliferation (6), whereas in the kidney, stimulating the receptor reduces renal tubular Ca2+ reabsorption (20). Physiological proof of the CaR's key roles in [Ca2+]o homeostasis has come from the identification of hyper- and hypocalcemic disorders caused by inactivating or activating CaR mutations (5), respectively, and from mice with targeted disruption of the CaR gene (23).
In addition to inhibiting PTH release from parathyroid cells, the CaR
stimulates the secretion of calcitonin from C cells (12,
14) and of ACTH from AtT-20 cells (11).
Furthermore, several studies have shown that high
[Ca2+]o can stimulate PTHrP release from
normal keratinocytes (22), normal cervical epithelial
cells (28), oral squamous cancer cells (31),
and JEG-3 cells (21), suggesting that the CaR could be the
mediator of high [Ca2+]o-evoked PTHrP release
from both normal and malignant cells. In the case of PTHrP-secreting
prostate cancers metastatic to bone, this CaR-mediated action could
create an inappropriate "feed-forward" stimulation of PTHrP
secretion, causing release of Ca2+ from bone that would
stimulate further PTHrP secretion and promote worsening bone
resorption. Moreover, interrupting high
[Ca2+]o-evoked, CaR-mediated PTHrP secretion
from prostate cancer cells [e.g., with a CaR antagonist
(15)] could potentially be of substantial clinical
benefit in this setting. The goals of the present study, therefore,
were to determine whether two commonly employed prostate cancer cell
lines, LnCaP and PC-3, express the CaR, and if so, whether this
receptor participates in the regulation of PTHrP secretion. Our results
suggest that the CaR is expressed in and likely mediates high
[Ca2+]o-induced PTHrP secretion from PC-3
cells. Furthermore, transforming growth factor
(TGF)-1, stimulates PTHrP secretion from PC-3
cells synergistically with high [Ca2+]o,
suggesting that release of this growth factor, along with calcium,
during PTHrP-induced bone resorption could contribute to a feed-forward
mechanism in which PTHrP-mediated osteolysis associated with prostate
cancers metastatic to bone begets worsening osteolysis.
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MATERIALS AND METHODS |
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Cell culture. The LnCaP and PC-3 human prostate cancer cell lines were obtained from the American Type Culture Collection (Rockville, MD). The cells were cultured in RPMI-1640 medium supplemented with 10% FCS and 100 U/ml penicillin-100 µg/ml streptomycin. The cells were grown at 37°C in a humidified 5% CO2 atmosphere and were passaged every 5-7 days with the use of either 0.25% trypsin-0.53 mM EDTA (LnCaP cells) or 0.05% trypsin-0.53 mM EDTA (PC-3 cells). All cell culture reagents were purchased from GIBCO-BRL (Grand Island, NY), with the exception of FCS, which was obtained from Gemini Bio-Products, (Calabasas, CA).
Northern blotting. Total RNA was prepared using TRIzol reagent (GIBCO-BRL). Northern blot analysis was performed on 7.5 µg of poly(A+) RNA obtained using oligo-dT cellulose chromatography of total RNA (8). Poly(A+)-enriched RNA samples were denatured and electrophoresed in 2.2 M formaldehyde-1% agarose gels along with a 0.24- to 9.5-kb RNA ladder (GIBCO-BRL) and transferred overnight to nylon membranes (Duralon; Stratagene, La Jolla, CA). A 32P-labeled riboprobe corresponding to nucleotides 1745-2230 of the human parathyroid CaR cDNA was synthesized with the MAXIscript T3 kit (Pharmacia Biotech, Piscataway, NJ) with the use of T3 RNA polymerase and [32P]UTP. Nylon membranes were then prehybridized, hybridized overnight with the labeled cRNA probe (2 × 106 cpm/ml), and washed at high stringency for 30 min as described previously (35). Membranes were sealed in plastic bags and exposed to a PhosphorImager screen. The screens were analyzed on a Molecular Dynamics PhosphorImager (Sunnyvale, CA) with the ImageQuant program.
RT-PCR. Total RNA (3-5 µg) was used for the synthesis of first-strand cDNA (cDNA synthesis kit, GIBCO-BRL). The resultant first-strand cDNA was used for PCR, which was performed in a buffer containing (in mM): 20 Tris · HCl, pH 8.4, 50 KCl, 1.8 MgCl2, and 0.2 dNTP and 0.4 µM forward primer, 0.4 µM reverse primer, and 1 µl ELONGASE enzyme mix (a Taq/Pyrococcus species GB-D DNA polymerase mixture; GIBCO-BRL). Human parathyroid CaR sense primer 5'-CGGGGTACCTTAAGCACCTACGGCATCTAA-3' and antisense primer 5'-GCTCTAGAGTTAACGCGATCCCAAAGGGCTC-3', which are intron spanning, were used for the reactions. To perform "hot start" PCR, the enzyme mixture was added during the initial 3-min denaturation and was followed by 35 cycles of amplification (30-s denaturation at 94°C, 30-s annealing at 47°C, and 1-min extension at 72°C). The reaction was completed with an additional 10-min incubation at 72°C to allow completion of extension. PCR products were fractionated on 1.5% agarose gels. PCR products in the reaction mixture were purified using the QIAquick PCR purification kit (Qiagen, Santa Clarita, CA) and were subjected to bidirectional sequencing by employing the same primer pairs used for PCR by means of an automated sequencer (AB377; Applied Biosystems, Foster City, CA) as previously described (35).
Immunocytochemistry. A CaR-specific polyclonal antiserum (4637) was generously provided by Drs. Forrest Fuller and Karen Krapcho of NPS Pharmaceuticals. This antiserum was raised against a peptide corresponding to amino acids 345-359 of the bovine CaR, which is identical to the corresponding peptide in the human CaR and resides within the predicted amino-terminal extracellular domain of the CaR. The antiserum was subjected to further purification by means of an affinity column conjugated with the FF-7 peptide (27), and the affinity-purified antiserum was used for immunocytochemistry and Western blot analysis as described in the following paragraphs. The specificity of the antiserum for the CaR is documented in RESULTS by the use of suitable positive and negative controls.
For immunocytochemistry, prostate cancer cells were grown on glass coverslips (27), fixed for 5 min with 4% formaldehyde, and then treated for 10 min with peroxidase blocking reagent (DAKO, Carpenteria, CA) to inhibit endogenous peroxidases. After washing with PBS, the cells were blocked for 30 min with PBS containing 1% BSA. The cells were then incubated overnight at 4°C with the 4637 antiserum (5 µg/ml in blocking solution). Negative controls were carried out by incubating cells treated in an otherwise identical manner with the same concentration of 4637 antiserum that had been preabsorbed with 10 µg/ml of the FF-7 peptide. The cells were then washed, incubated with peroxidase-conjugated goat anti-rabbit IgG (1:100; Sigma Chemical, St. Louis, MO) and washed again, and the color reaction was developed using the DAKO AEC substrate system (DAKO) as before (27). The cells were observed by light microscopy and photographed at ×400 magnification.Western Blotting.
For Western blotting, confluent monolayers of LnCaP and PC-3 cells in
6-well plates were rinsed with ice-cold PBS and scraped on ice into
lysis buffer containing 10 mM Tris · HCl, pH 7.4, 1 mM EGTA, 1 mM EDTA, 0.25 M sucrose, 1% Triton X-100, 1 mM dithiothreitol, and a
cocktail of protease inhibitors (10 µg/ml each of aprotinin, leupeptin, and calpain inhibitor, as well as 100 µg/ml of Pefabloc) (26). The cells were then passed though a 22-gauge
needle 10 times. Nuclei and other cellular debris were removed by
low-speed centrifugation (1,000 g for 10 min), and the
resultant total cellular lysate in the supernatant was used either
directly for SDS-PAGE or stored at 80°C. Bovine parathyroid cells,
CaR-transfected HEK-293 cells (designated HEKCaR), or nontransfected
HEK-293 cells, included as positive (parathyroid and HEKCaR) and
negative controls (nontransfected HEK-293 cells), were harvested
according to the same protocol.
Adenoviral infection of dominant negative CaR into PC-3 cells. Confluent PC-3 cells were scraped, dispersed by repeated pipetting, and then seeded in 24-well plates (~2.5 × 103 cells/well). Approximately 10,000 infective particles containing dominant negative CaR (R185Q) or empty vector as a negative control were added to each well at the time the cells were seeded in growth medium. The cells were then cultured for 48 h, washed with PBS, and then incubated with DMEM (containing 0.2% BSA and 0.5 mM [Ca2+]o) for 2 h. Additional calcium was then added to the wells as needed to achieve the final concentrations indicated in RESULTS, and the cells were incubated overnight. At the end of the incubation, conditioned medium was collected and subjected to PTHrP assay as described in PTHrP secretion studies. The data were normalized to the amount of protein in each well. Experiments were carried out using triplicate wells for each level of [Ca2+]o.
PTHrP secretion studies. For studies on the effects of various CaR agonists on PTHrP secretion, PC-3 cells were seeded in 96-well plates (5,000 cells/well) in 0.15 ml of medium A (RPMI-1640 supplemented with 10% FCS and 100 U/ml penicillin-100 µg/ml streptomycin). After 72 h, medium A was carefully removed, and the subconfluent cells in each well were rinsed once with 0.15 ml of medium B [calcium-free DMEM (GIBCO-BRL) supplemented with 4 mM L-glutamine, 2% FCS, 100 U/ml penicillin-100 µg/ml streptomycin, and 0.5 mM CaCl2]. Medium B alone or medium B supplemented with either additional CaCl2 (to final concentrations of 1, 3, 5, 7.5, or 10 mM) or the polycationic CaR agonists neomycin (100 or 300 µM) or spermine (2 mM) was then added to each well (0.275 ml/well). Six hours later, the conditioned medium was removed for determination of PTHrP content. Triplicate incubations were performed for each treatment, and each experiment was carried out at least twice.
For studies on the effects of pretreatment with TGF-Statistical analyses. A minimum of two independent PTHrP secretion experiments were performed for each of the PTHrP secretion studies described earlier. Results are presented as means ± SE for three determinations. Data were analyzed by analysis of variance followed by Fisher's protected least significant difference test. For all statistical tests, a P value <0.05 was considered to indicate a statistically significant result.
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RESULTS |
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Detection of CaR mRNA in
LnCaP and PC-3 cells by Northern analysis
and RT-PCR.
Northern blot analysis carried out using a CaR-specific riboprobe on
poly(A+) RNA isolated from LnCaP and PC-3 cells revealed a
major transcript of ~5.2 kb (Fig.
1A). This transcript is
similar in size to a major CaR transcript in human parathyroid gland
(13). RT-PCR performed with intron-spanning primers
specific for the human CaR amplified a product of the expected size,
480 bp, for a CaR-derived product in both LnCaP (Fig. 1B,
lane 2) and PC-3 cells (Fig. 1B, lane
3). DNA sequence analysis of the PCR products revealed >99% sequence identity with the corresponding region of the human
parathyroid CaR cDNA (not shown). These results indicate that the PCR
products derived from both PC-3 and LnCaP cells were amplified from
authentic CaR transcript(s).
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Detection of CaR protein in LnCaP and
PC-3 cells by immunocytochemistry and Western analysis.
Immunocytochemistry with an anti-CaR antiserum (4637) revealed moderate
CaR staining in both LnCaP (Fig.
2A) and PC-3 (Fig. 2B) prostate cancer cells. Staining was eliminated by
preincubating the CaR antiserum with the specific peptide (FF-7)
against which it was raised (Fig. 2, C and D).
Considerable intracellular CaR immunoreactivity could be observed in
these cells, as in breast cancer (35) and bone cells
(43, 44), which express considerably less CaR protein than
do parathyroid cells (26), where the CaR displays a
predominantly rim-like pattern of cell surface expression. Western blot
analyses of proteins isolated from total cellular lysates of LnCaP or
PC-3 cells by use of the 4637 antiserum were compared with those
obtained using protein preparations from HEKCaR and bovine parathyroid
cells as positive controls and nontransfected HEK-293 cells as a
negative control (Fig. 3, A
and C). Although the level of CaR protein expression in
HEKCaR cells was much higher than the level in LnCaP and PC-3 cells
(Fig. 3A), the immunoreactive bands in the two prostate
cancer cell lines of ~160-170 kDa are comparable in size to
those of bands present in the positive controls (Fig. 3, A
and C). The specificity of these 160- to 170-kDa
CaR-immunoreactive bands in proteins from the prostate cancer cell
lines was confirmed by the marked reductions in their intensities after
preabsorption of the antiserum with the peptide against which it was
raised, although nonspecific bands at lower molecular masses were not abolished by the preabsorption procedure (Fig. 3B).
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Effect of CaR agonists,
TGF-1, and dominant negative
CaR on PTHrP secretion.
To determine whether CaR agonists modulate PTHrP secretion from PC-3
cells, the cells were treated with varying levels of [Ca2+]o (0.5, 1, 3, 5, 7.5, or 10 mM),
neomycin (100 or 300 µM in 0.5 mM [Ca2+]o),
or spermine (2 mM in 0.5 mM [Ca2+]o), and
PTHrP in the conditioned medium was determined by IRMA. PC-3 cells
produce a readily measurable amount of PTHrP at 0.5 mM
[Ca2+]o. Higher levels of
[Ca2+]o stimulated PTHrP secretion in
a dose-dependent manner (Fig. 4A). At 1, 3, and 5 mM
[Ca2+]o, PTHrP secretion was increased 1.2-, 1.5-, and 1.8-fold, respectively, compared with that observed at 0.5 mM
[Ca2+]o. [Ca2+]o at
7.5 and 10 mM evoked more substantial increases in PTHrP secretion
(3.0- and 3.2-fold, respectively). The polycationic CaR agonists
neomycin and spermine also elicited robust secretory responses: 100 and
300 µM neomycin increased PTHrP secretion 3.4- and 3.6-fold,
respectively, relative to that at 0.5 mM
[Ca2+]o, whereas 2 mM spermine induced a
4.6-fold increase in secretion.
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DISCUSSION |
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The purpose of this study was to determine whether the LnCaP and PC-3 human prostate cancer cell lines express the CaR, and if so, whether CaR agonists modulate PTHrP secretion from them. CaR expression was detected in LnCaP and PC-3 cells by both nucleotide- and protein-based approaches. Northern analysis performed on poly(A+) RNA from each of the two cell lines revealed a 5.2-kb CaR transcript (Fig. 1A). This transcript is similar in size to one of the predominant CaR transcripts observed in human parathyroid cells (13). Authentic CaR transcript(s) was also detected by RT-PCR (Fig. 1B), performed using total RNA from LnCaP and PC-3 cells followed by sequence analysis of the PCR products.
These two prostate cancer cell lines also express CaR protein as assessed by immunocytochemistry (Fig. 2) and Western blot analysis (Fig. 3) performed using an affinity-purified, anti-CaR antiserum (4637). As assessed by Western analysis, the levels of CaR protein expression in LnCaP and PC-3 cells were substantially lower than in the positive controls, HEKCaR cells and bovine parathyroid cells. They are not dissimilar, however, from those in several other types of cells in which we have shown that the CaR is expressed and modulates various biological responses, such as regulation of Ca2+-activated K+ channels (9).
[Ca2+]o and the polycationic CaR agonists neomycin and spermine each stimulated PTHrP secretion from LnCaP and PC-3 cells in a dose-dependent manner (Fig. 3A), with maximal stimulation occurring at 7.5-10 mM [Ca2+]o. The levels of [Ca2+]o in the vicinity of resorbing osteoclasts are thought to be many times higher than the level of systemic [Ca2+]o (i.e., as high as 8-40 mM) (36). Therefore, in the bony microenvironment, metastatic prostate cancer cells will likely encounter levels of [Ca2+]o at least as high as those used in the present studies. Our results are consistent with those in other cell types exhibiting high [Ca2+]o-evoked PTHrP secretion, including normal keratinocytes (22), normal cervical epithelial cells (28), oral squamous cancer cells (31), JEG-3 cells (21), and H-500 rat Leydig cells, a model of humoral hypercalcemia of malignancy (34). The molecular mechanism underlying [Ca2+]o-stimulated PTHrP secretion in these cell types, however, is not clear. Our data suggest that the CaR is the likely mediator of this effect in PC-3 cells, because the receptor is clearly expressed in this cell line and PTHrP secretion is stimulated not only by elevated levels of [Ca2+]o but also by the polycationic CaR agonists neomycin and spermine. Furthermore, adenovirus-mediated infection of the PC-3 cells with a dominant negative CaR (R185Q) (2) attenuated and right-shifted high [Ca2+]o-stimulated PTHrP secretion, providing additional strong evidence for mediation of this action by the CaR. Others have successfully utilized transfection of CaR-expressing cells with a different dominant negative CaR construct (R795W) to document the CaR's involvement in other biological responses (30).
On the basis of the present study on PC-3 cells and in two breast
cancer cell lines (35), our findings have clear
implications for the existence of a feed-forward mechanism involving
prostate cancer cells metastatic to bone. When prostate and breast, and possibly other, cancers metastasize to the skeleton and induce PTHrP-mediated osteolysis, this will lead to high local levels of
[Ca2+]o within the bony microenvironment
owing to PTHrP-stimulated bone resorption with or without associated
systemic hypercalcemia. These high levels of
[Ca2+]o will elicit further PTHrP secretion
from the cancer cells, thereby exacerbating the osteolytic disease.
Guise and Mundy (18) have provided strong evidence for the
existence of a similar feed-forward mechanism involving the action of
TGF- released from bone on PTHrP-secreting breast cancer cells.
Indeed, we have shown that TGF-
1 increases PTHrP
secretion from PC-3 cells and have also demonstrated that
TGF-
1 produces at least an additive increase in the
stimulation of PTHrP secretion by high
[Ca2+]o. The mechanism for this effect is not
clear but might involve TGF-
1-induced upregulation of
the expression of the CaR or its signaling pathways and/or of the level
of expression of the PTHrP gene, thereby increasing the amount of PTHrP
available for secretion in response to an elevated level of
[Ca2+]o. Because
[Ca2+]o and TGF-
are both released from
the bone matrix during bone resorption induced by PTHrP, they are both
available to elicit further PTHrP secretion. In effect, both could
cooperate to generate a vicious cycle of tumor-induced bone resorption
begetting further bone resorption in the setting of skeletal metastases
of prostate (or breast) cancers. The beneficial actions of
bisphosphonates on the skeletal complications of metastatic breast
cancer and on the incidence of new metastases (17, 32, 41)
could result, at least in part, from reductions in the local
concentrations of both [Ca2+]o and TGF-
as
a result of decreased bone resorption.
In addition to its potential role in stimulating PTHrP secretion from prostate cancer cells metastatic to bone, the CaR could also impact on tumor progression, osteolysis, and, in some cases, hypercalcemia by modulating the proliferation and/or apoptosis of tumor cells. Recent studies have shown that CaR activation stimulates proliferation in several cell types, including rat-1 fibroblasts (30). In PTHrP-producing tumors, the CaR could potentially increase proliferation directly and/or indirectly by enhancing PTHrP secretion. Indeed, PTHrP has been shown to stimulate the proliferation of H-500 rat Leydig cells in vitro and to increase the rate of tumor growth in vivo when H-500 cells are implanted subcutaneously in rats (33). The CaR also protects some cells against apoptosis, as we have shown recently for AT-3 rat prostate cancer cells and CaR-transfected, but not nontransfected, HEK-293 cells (29). Therefore, high [Ca2+]o-evoked, CaR-mediated stimulation of proliferation and/or inhibition of apoptosis of prostate cancer cells metastatic to bone could clearly contribute to the progression of tumor growth and potentially render the tumor cells resistant to therapy.
In summary, high [Ca2+]o-evoked, CaR-mediated PTHrP secretion could clearly contribute to the excessive bone resorption recently recognized to be an important complication of prostate cancer metastatic to bone. If, as in PC-3 cells, the CaR modulates PTHrP secretion in other prostate cancer cells, then the use of CaR antagonists (15) with some degree of specificity for prostate and other types of cancer cells that metastasize to bone and produce PTHrP and, therefore, osteolysis could potentially offer substantial therapeutic benefits in this setting.
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ACKNOWLEDGEMENTS |
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Generous grant support for this work was provided by the National Institute of Diabetes and Digestive and Kidney Diseases (DK-09835 to J. L. Sanders and DK-48330 to E. M. Brown), NPS Pharmaceuticals, and the St. Giles Foundation (to E. M. Brown).
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FOOTNOTES |
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* These authors contributed equally to this work.
Address for reprint requests and other correspondence: E. M. Brown, Endocrine-Hypertension Division, Brigham and Women's Hospital, 221 Longwood Ave., Boston, MA 02115.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 26 September 2000; accepted in final form 11 July 2001.
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