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St. Vincent’s Institute of Medical Research and The University of Melbourne, Department of Medicine (R.J.T., J.E., N.J.H., T.J.M., M.T.G.) St. Vincent’s Hospital, Fitzroy, Victoria 3065, Australia; and The Department of Medicine, Division of Endocrinology at the University of Texas Health Science Center, (T.A.G., J.J.Y.) San Antonio, Texas 78284
Address all correspondence and requests for reprints to: Dr. Matthew T. Gillespie, St. Vincent’s Institute of Medical Research, 41 Victoria Parade, Fitzroy 3065, Victoria, Australia. E-mail: m.gillespie{at}medicine.unimelb.edu.au
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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B ligand), expressed by osteoblasts has
been cloned as well as its cognate signaling receptor, receptor
activator of NFB (RANK), and a secreted decoy receptor
osteoprotegerin (OPG) that limits RANKL’s biological action. We
determined that the breast cancer cell lines MDA-MB-231, MCF-7, and
T47D as well as primary breast cancers do not express RANKL but express
OPG and RANK. MCF-7, MDA-MB-231, and T47D cells did not act as
surrogate osteoblasts to support osteoclast formation in coculture
experiments, a result consistent with the fact that they do not express
RANKL. When MCF-7 cells overexpressing PTH-related protein (PTHrP) were
added to cocultures of murine osteoblasts and hematopoietic cells,
osteoclast formation resulted without the addition of any osteotropic
agents; cocultures with MCF-7 or MCF-7 cells transfected with pcDNAIneo
required exogenous agents for osteoclast formation. When MCF-7 cells
overexpressing PTHrP were cultured with murine osteoblasts,
osteoblastic RANKL messenger RNA (mRNA) levels were enhanced and
osteoblastic OPG mRNA levels diminished; MCF-7 parental cells had no
effect on RANKL or OPG mRNA levels when cultured with osteoblastic
cells. Using a murine model of breast cancer metastasis to bone, we
established that MCF-7 cells that overexpress PTHrP caused
significantly more bone metastases, which were associated with
increased osteoclast formation, elevated plasma PTHrP concentrations
and hypercalcaemia compared with parental or empty vector controls. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Most breast cancer metastases in bone form osteolytic lesions, in
contrast with bone secondaries from prostate cancer which are
osteosclerotic (1). Although it has been postulated that bone
destruction by breast cancer is mediated directly by tumor cells (1),
most evidence indicate that breast cancer-induced bone destruction is
mediated by the osteoclast. Support for the latter include 1) breast
cancers express cytokines (such as IL-1, IL-6, LIF, prostaglandin
E2 (PGE2), tumor necrosis factor-
(TNF)
and PTHrP) which can influence osteoclast formation (1); 2) histologic
analysis of osteolytic lesions reveal tumor adjacent to osteoclasts
resorbing bone; 3) and use of bisphosphonates, potent inhibitors of
osteoclastic bone resorption, in women with breast cancer metastases to
bone results in reduced skeletal morbidity (8, 9).
The process of mouse osteoclast formation can be studied in vitro by culturing bone marrow culture cells and by coculture of osteoblastic stromal cells with hematopoietic cells, both of which result in the formation of bona vide osteoclasts in response to various osteotropic factors; the osteoclasts stain for tartrate-resistant acid phosphatase (TRAP), are multinucleated, exhibit calcitonin receptors (CTR) and most importantly can resorb bone (10, 11). In both systems, stromal osteoblasts support osteoclast formation from precursors of hematopoietic origin.
Recently the stromal cell-derived osteoclast differentiation factor
(ODF) or osteoprotegerin ligand (OPGL) has been identified, and a
soluble form of the molecule in combination with M-CSF can generate
osteoclasts from hematopoietic cells in the absence of osteoblastic
stromal cells (11, 12, 13, 14): it was also identified by its ability to induce
NFB and apoptosis of T-cells as receptor activator of NF-B ligand
(RANKL) and tumor necrosis factor-related activation-induced cytokine
(TRANCE), respectively (15, 16, 17). For simplicity, will we adopt its
nomenclature as RANKL, which represents the functional property that
has been described for this molecule. RANKL is a member of the tumor
necrosis factor (TNF) family and is a membrane-bound molecule, there is
no evidence for an alternatively spliced form of this molecule,
although the molecule is shed from the plasma membrane as a result of
protease action from HEK293 cells overexpressing RANKL/OPGL (12). Two
receptors for RANKL have been proposed. The first, which aided the
identification RANKL was osteoprotegerin (OPG), also reported as
osteoclastogenesis inhibitory factor (OCIF) (18, 19). OPG is a secreted
TNF receptor family member and has a relatively wide distribution.
Overexpression of OPG in mice resulted in osteopetrosis (18).
Conversely, mice deficient in OPG demonstrate osteoporosis and
calcification of the aorta (20). In agreement with the phenotypes of
mice with altered OPG production, recombinant OPG inhibited osteoclast
formation in cocultures of mouse osteoblastic cells and hematopoietic
cells (12, 13). The ability of OPG to bind to RANKL and limit the
biological actions of RANKL suggested that OPG may function as a decoy
receptor (12, 13, 19, 21). The second, putative receptor, and likely
responsible for signaling RANKL biological actions, appears to be
receptor activator of NFB (RANK) (15), although other hitherto
unrecognized receptors of the TNF-receptor family may have similar
capabilities. In this study, we have investigated the expression of
RANKL, OPG, and RANK in breast cancer cell lines and primary tumors. We
have also investigated whether breast cancers can directly support or
indirectly influence osteoclastogenesis using the murine coculture
system and we have demonstrated the ability of breast cancer cells to
regulate RANKL expression of stromal osteoblasts. Finally, we have
assessed the in vivo osteolytic potential of breast cancer
cell lines which differ in their osteoclast-inductive capacity in
vitro.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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,25(OH)2 D3 was purchased from Wako Pure Chemical Co. (Osaka, Japan), and PGE2 was
obtained from Sigma (St. Louis, MO).
Coculture system
Osteoblastic cells were prepared from the calvaria of newborn
mice by sequential digestion with 0.1% collagenase (Worthington Biochemical Corp. Co., Freefold, Australia) and 0.2% dispase
(Godo Shusei, Tokyo, Japan). Bone marrow was obtained from the femur
and tibia of adult C57BL6J male mice. Osteoblastic cells and/or breast
cancer cells were cocultured with spleen or marrow cells as previously
described (22, 23). The expression of calcitonin receptors was also
assessed by autoradiography using [125I]-salmon
calcitonin as described (22) and resultant cells from coculture
experiments were tested for their ability to resorb bone (24).
Tissue specimens
Twelve breast lesions were collected from patients undergoing
resective surgery at St. Vincent’s Hospital. The tissues were
immediately placed on dry ice and stored at -70 C. The tissues
examined were all infiltrating ductal carcinomas with two tumors
containing a ductal carcinoma in situ component.
RNA extraction, complementary DNA (cDNA) synthesis, and PCR
Total RNA extraction from the tissues, cDNA synthesis, and PCR
were performed as described (25). Oligonucleotides were designed to
amplify and detect human RANKL (GenBank accession number AF019047),
murine RANKL (GenBank accession number AF019048), human OPG (GenBank
accession number U94332), murine OPG (GenBank accession number U94331)
and human RANK (GenBank accession number AF018253) (Table 1
). Finally, for glyceraldehyde phosphate
dehydrogenase (GAPDH) primers used have been published previously
GAPDH-1, GAPDH-2, GAPDH-3, and GAPDH-4 (25). The specificity of the
products was confirmed by Southern blot detection using a
32P-labeled internal oligonucleotide probe, as previously
described (25) or by nucleic acid sequencing of amplified products.
Bound probe was detected by PhosphorImager analysis (Molecular Dynamics, Inc., Sunnyvale, CA).
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In vivo experiments
Animal protocols were approved by the Institutional Animal Care
and Use Committee at the University of Texas Health Science Center at
San Antonio and were in accordance with the NIH Guide for the Care and
Use of Laboratory Animals. Female nude mice 4–6 weeks of age were
housed in laminar flow isolated hoods with 12-h light, 12-h dark cycle.
Water supplemented with vitamin K and autoclaved mouse chow were
provided ad libitum.
Whole blood samples for ionized calcium concentration were obtained by retro-orbital puncture under metofane anesthesia. Blood samples for PTHrP measurement were similarly obtained and collected on ice in vacutainer tubes containing EDTA (Becton Dickinson and Co., NJ) and 400 IU/ml aprotinin (Sigma, St. Louis, MO).
Tumor inoculation into the left cardiac ventricle was performed while the mice were anesthetized with a ketamine/xylazine mixture and positioned ventral side up based on a modification of Arguello (27). Because MCF-7 cells are estrogen dependent, mice were implanted with a 60-day slow release 17ß-estradiol pellet (0.5 mg, 3 mm; Innovative Research of America, Sarasota, FL) before tumor inoculation. The left cardiac ventricle was punctured percutaneously using a 27-gauge needle attached to a 1-ml syringe containing suspended tumor cells. Visualization of bright red blood entering the hub of the needle in a pulsatile fashion indicated correct position in the left cardiac ventricle.
Bone metastasis
Mice were inoculated with tumor cell suspensions of MCF-7 PTHrP
139c, MCF-7 pcDNAIneo or MCF-7 cells into the left cardiac ventricle
(n = 5 per group) on day 0. Baseline radiographs and body weights
as well as blood for Ca2+ and plasma PTHrP concentrations
were obtained at this time. Radiographs were taken on day 21 and then
weekly until they were killed. At the time mice were killed, blood was
collected for Ca2+ and PTHrP measurement, and all bones and
soft tissues were harvested and fixed in formalin for histologic
analysis. Autopsy was performed on all mice, and those with tumor in
the chest were excluded from analysis, as this indicated that part or
all of the tumor inoculum did not properly enter the left cardiac
ventricle. This experiment was performed twice with similar results
obtained.
Ca2+ measurement
Ca2+ concentrations were measured in whole blood
using a Ciba Corning, Inc. 634 ISE Ca2+/pH
analyzer (Medfield, MA) and adjusted using the internal algorithm of
the instrument to pH 7.4. Samples were run in duplicate and the mean
value recorded.
PTHrP assay
PTHrP concentrations were measured in conditioned media and in
plasma using a two-site immunoradiometric assay (Nichols Institute Diagnostics, San Juan Capistrano, CA), which uses two
polyclonal antibodies that are specific for the
N-terminal-(1–40) and-(60–72) portions of PTHrP and has a
calculated sensitivity of 0.3 pmol/liter (28).
PTHrP concentrations in conditioned media samples were calculated from a standard curve generated by adding recombinant PTHrP (1–86) to the specific type of medium (unconditioned) used and were considered undetectable if media concentrations were < 0.3 pmol/liter before correction for cell number.
Radiographs and measurement of osteolytic lesion area
Animals were x-rayed in a prone position against the film
(22 x 27 cm X-Omat AR, Eastman Kodak Co., Rochester,
NY) and exposed with x-rays at 35 KVP for 6 sec using a Cabinet x-ray
system-Faxitron Series, Hewlett-Packard Co. (Model 43855A;
Faxitron X-Ray Corp., Buffalo Grove, IL). All radiographs were
evaluated in blinded fashion. The area of osteolytic bone metastases
was calculated using a computerized image analysis system. Video images
of radiographs were captured using a frame grabber board
(Targa+, Truevision, Inc.) on a PC system.
Quantitation of lesion area was performed using image analysis software
(Java, Jandal Video analysis, Jandel Scientific, CA).
Bone histology and histomorphometry
Fore- and hindlimb bones were removed from mice at time of
killing, fixed in 10% buffered formalin, decalcified in 14% EDTA, and
embedded in paraffin wax. Sections were cut using a standard microtome,
placed on poly-L-lysine-coated glass slides and stained
with hematoxylin, eosin, orange G and phloxine.
The following variables were measured in midsections of tibiae and femora, without knowledge of treatment groups, to assess tumor involvement: total tumor area and osteoclast number per millimeter of tumor/bone interface. Histomorphometic analysis was performed on an OsteoMeasure System (Osteometrics, Atlanta,GA).
Statistical analysis
Results are expressed as the mean ± the SEM.
Data were analyzed by repeated measures ANOVA followed by Turkey-Kramer
post test. P values of < 0.05 were considered
significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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,25(OH)2
D3 and PGE2, for 7 days osteoclasts were formed
(TRAP+, MNC, exhibiting CTR and capable of resorbing bone)
(Fig. 1A). In contrast, none of the
breast cancer lines MDA-MB-231, T47D, MCF-7, and MCF-7 PTHrP 139c were
able to support osteoclast formation when cocultured with hematopoietic
cells from neonatal mouse spleen or adult mouse marrow in the presence
of 1,25(OH)2 D3 and PGE2 (Fig. 1A). This indicated that breast cancer cell lines alone were unable to
substitute for stromal osteoblasts. No multinucleated osteoclasts were
formed when marrow or spleen cells were incubated alone even in the
presence of 1,25(OH)2D3 and PGE2
as has been previously described (29).
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) or neonatal mouse spleen (data not shown). These models are more
akin to the in vivo situation, whereby breast cancer cells
interact with both osteoblasts and hematopoietic cells. When
osteoblastic cells were cultured with hematopoietic cells, osteoclasts
were formed in response to treatment with 1,25(OH)2
D3 and PGE2. Similarly, when any of the breast
cancer cell lines, MCF-7 (Fig. 1B), MDA-MB-231 or T47D (not shown),
were added in equal numbers to the osteoblastic cells (5 x
104 cells/48-well culture plate), osteoclast formation
occurred and the resultant osteoclast numbers were similar to that seen
with osteoblast and hematopoietic cell cocultures (Fig. 1B). The small
discrepency in osteoclast numbers seen between the osteoblastic
coculture and the MCF-7 coculture maybe due to the high level of
expression of OPG seen in the MCF-7 cells (Fig. 2A), which could diminish osteoclast
formation.
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).
These cells displayed the properties of osteoclasts i.e.
TRAP+, multinucleated, expressed CTR and could resorb bone.
Upon addition of 1,25(OH)2 D3 and
PGE2 osteoclast formation was equivalent to that generated
by osteoblasts with these treatments (Fig. 1B). The resultant
osteoclast formation seen with the PTHrP-overexpressing cells in the
absence of any osteotropic agents contrasts markedly to that of the
parental MCF-7 cell line. Because the TNF-related ligand (RANKL) and the receptors (OPG, RANK) have been invoked as pivotal molecules in osteoclast formation (21), we determined the expression of RANKL, OPG, and RANK mRNA in breast cancer cell lines and in primary breast cancers. Total RNA was isolated and reverse transcribed then subjected to PCR for 40 cycles of amplification, which represent saturating, nonquantitative, conditions. As controls, RT-PCR was performed on RNA isolated from a giant cell tumor of bone (GCT), which we have determined to express RANKL, OPG, and RANK. As a negative control, PCRs were performed on RNA, which had not been reversed transcribed and no products were detected under these conditions.
Consistent with the inability of breast cancer cell lines to support
osteoclast formation, the lines MDA-MB-231, T47D, MCF-7, and MCF-7
PTHrP 139c did not express RANKL mRNA, whereas the GCT expressed a
single product with nucleotide sequence identity to RANKL (Fig. 2A).
Additionally, none of the primary breast cancers expressed RANKL mRNA
(Fig. 2A). Because RNA from the primary tumors would represent RNA from
both cancer cells and from the normal stroma, this implies that both
the stroma and breast cancer cells fail to express RANKL. In contrast,
the three breast cancer cell lines and all tumors expressed mRNA for
OPG and RANK (Fig. 2A); albeit that OPG levels were lower in the T47D
cells relative to the MCF-7 and MDA-MB-231 cells as has been reported
previously (31). The MCF-7 PTHrP 139c cells showed an equivalent level
of OPG mRNA expression to the parental MCF-7 cells (data not shown).
Using the GAPDH primers, a product of the predicted size was amplified
from each of the tumor and cell line samples, which hybridized with the
internal GAPDH oligonucleotide, GAPDH-1 (Fig. 2A), thus confirming the
integrity of RNA used.
RANKL mRNA is regulated by osteotropic factors in osteoblasts (13, 32)
and its expression relative to that of OPG expression appears to
dictate the osteoclast-inductive nature of cells (32). Thus, we
examined the effect of local and systemic factors such as IL-11, PTHrP,
transforming growth factor ß (TGFß) and 1,25(OH)2
D3 to stimulate the expression of RANKL in the MDA-MB-231,
T47D, and MCF-7 cell lines. However, none of these agents permitted the
expression of RANKL in any of these cell lines (data not shown),
suggesting that the RANKL gene in breast cancers is
maintained in a transcriptionally inactive state. Such a result is in
accordance with the inability of breast cancer cell lines to act in a
manner equivalent to osteoblastic stromal cells for differentiation and
induction of osteoclasts from hematopoietic cells.
PTHrP expressed by breast cancers modulates osteoblast RANKL
production
We have previously established that osteoblastic RANKL mRNA levels
can be enhanced by PTH/PTHrP (32). To determine if PTHrP produced by
breast cancer cells invoked changes in RANKL mRNA similar to those we
observed in response to treatment of primary osteoblasts, murine RANKL
and OPG mRNA levels were assessed in cocultures of human breast cell
lines and primary murine osteoblasts (Fig. 2B). MCF-7 parental and the
PTHrP overexpressing cell line, MCF-7 PTHrP 139c, were cultured with
primary mouse osteoblasts (osteoblasts at 1 x 106
cells and MCF-7 at 5 x 105 cells per 10 mm Petri
dish), and murine RANKL and OPG levels were determined during 7 days of
coculture. Mouse RANKL mRNA was induced 2.4 ± 0.2-fold following
8 h of coculture with MCF-7 PTHrP 139c cells and remained elevated
for up to 72 h. Levels of murine RANKL mRNA were unaltered in
cocultures comprising MCF-7 cells for up to 3 days, then in both MCF-7
and MCF-7 PTHrP 139 cocultures murine RANKL mRNA levels decreased by
day 7 (Fig. 2B). Furthermore, murine OPG mRNA expressed by osteoblastic
cells was reduced 6.1 ± 0.5-fold at 8 h when MCF-7 cells
overexpressing PTHrP were cocultured with murine osteoblasts compared
with parental MCF-7 cells.
In vivo experiments
Given that the MCF-7 cells overexpressing PTHrP enhanced
osteoclast formation in murine in vitro cocultures, we
sought to establish the role of PTHrP overexpression by the MCF-7 cell
line in vivo by intracardiac injection in the nude mouse
model. Such a model has demonstrated that MDA-MB-231 cells avidly
metastasize to bone and induce osteolysis (30).
Mice inoculated with the MCF-7 PTHrP 139c developed large bone
metastases with osteolysis being evident earlier and to a greater
extent than that seen with mice harbouring either the parental cells or
cells stably transfected with the vector control only (Fig. 3). When quantitated by computerized
image analysis of radiographs, the differences in lesion area and
number were statistically significant (Fig. 3). The in vitro
growth rates of these MCF-7 cells showed no significant difference
between the MCF-7 PTHrP-139 and the empty vector or parental cells
(data not shown) therefore the difference in tumor size is not
attributable to the growth rate of the various MCF-7 cells.
Histomorphometric analysis of bone (Fig. 4, A and B) indicate that osteoclast
number per mm of tumor-bone interface was markedly greater, as was
tumor area, in mice bearing MCF-7 PTHrP-139 tumors compared with
the control parental and empty vector groups. Significant hypercalcemia
was evident in mice bearing the MCF-7 PTHrP-139 cells, whereas mice
bearing the MCF-7 pcDNAIneo (empty vector) or
parental cells, remained normocalcemic (Fig. 4C). Concomitant with this
hypercalcemia, plasma PTHrP concentrations were significantly greater
at the time mice were killed (Fig. 4D) in the MCF-7 PTHrP-139c bearing
mice. There was no significant difference in body weight between mice
bearing tumors of MCF-7 PTHrP 139c, empty vector or parental cells (not
shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Several studies have implicated PTHrP as a crucial factor in the
process of breast cancer metastases in bone (2, 30). This study extends
these observations to demonstrate that PTHrP produced by breast cancer
cells is sufficient to stimulate osteoclast formation in marrow
cultures with osteoblastic cells without the requirement for exogenous
osteotropic agents such as 1,25 (OH)2 D3,
PGE2, or IL-11. In such cocultures we show the capacity of
breast cancer cell-derived PTHrP to induce osteoblastic RANKL mRNA
levels and reduce osteoblastic OPG mRNA levels, the net effect of which
is anticipated to enhance osteoclast formation. Such a finding is
concordant with the osteoclast induction seen when breast cancer cells
overexpressing PTHrP were added to cocultures and to previous in
vitro experiments where exogenous PTH/PTHrP similarly modulate
RANKL and OPG mRNA levels (32).
Finally, and most significantly, we extend these in vitro findings to an in vivo model of metastasis using the well established nude mouse model whereby cancer cells were administered via intracardiac injection. Whilst parental MCF-7 cells had a low prevalence for metastasis in bone, when PTHrP was overexpressed the cells avidly metastasized to bone and induced osteolysis with accompanying hypercalcemia. The MCF-7 cells have not been shown previously to be capable of metastatic growth in the nude mouse.
Combining the in vitro and in vivo data, a
possible model for the severe osteolysis induced by breast cancers is
proposed in Fig. 5. This model
extrapolates from our previous findings (2) and results presented
herein. As a consequence of breast cancer cells establishing in the
bone microenvironment, PTHrP secreted from these cells can act in a
paracrine/juxtacrine manner on osteoblastic cells, increasing RANKL
expression and limiting OPG expression. This favors the formation and
the survival of osteoclasts because RANKL has also been demonstrated to
limit osteoclast apoptosis (14). Enhancement of osteoclast numbers and
their activity results in pronounced osteolysis with the subsequent
release of bone-derived growth factors such as TGFß. TGFß is a
potent stimulator of PTHrP production acting both transcriptionally and
posttranscriptionally via mRNA stabilization (33, 34). Recently, TGFß
has been demonstrated to decrease RANKL mRNA and enhance OPG mRNA
levels in osteoblasts (35). Such a mechanism may well account for the
local control of bone formation and resorption. However, when breast
cancer cells have established in the bone environment, differential
roles of bone-derived growth factors, including TGFß, can emerge.
Thus for example, they could modify the production of cytokines and
growth factors by breast cancer cells and osteoblastic cells in ways
that could influence RANKL, OPG and the signaling receptor RANK.
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Footnotes |
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Received January 21, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, 25-dihydroxyvitamin D3 in osteoclast-like multinucleated cells
formed from mouse bone marrow cells. Endocrinology 123:1504–1510
-inducing factor) is produced by
osteoblasts and acts via granulocyte/macrophage colony-stimulating
factor and not via interferon-gamma to inhibit osteoclast formation. J
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G. van der Pluijm, I. Que, B. Sijmons, J. T. Buijs, C. W.G.M. Lowik, A. Wetterwald, G. N. Thalmann, S. E. Papapoulos, and M. G. Cecchini Interference with the Microenvironmental Support Impairs the De novo Formation of Bone Metastases In vivo Cancer Res., September 1, 2005; 65(17): 7682 - 7690. [Abstract] [Full Text] [PDF]
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 D. E. Godler, A. N. Stein, O. Bakharevski, M. M. L. Lindsay, and P. F. J. Ryan Parathyroid hormone-related peptide expression in rat collagen-induced arthritis Rheumatology, September 1, 2005; 44(9): 1122 - 1131. [Abstract] [Full Text] [PDF]
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N Normanno, A De Luca, D Aldinucci, M R Maiello, M Mancino, A D'Antonio, R De Filippi, and A Pinto Gefitinib inhibits the ability of human bone marrow stromal cells to induce osteoclast differentiation: implications for the pathogenesis and treatment of bone metastasis Endocr. Relat. Cancer, June 1, 2005; 12(2): 471 - 482. [Abstract] [Full Text] [PDF]
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A. H. Gordon, R. J. O'Keefe, E. M. Schwarz, R. N. Rosier, and J. E. Puzas Nuclear Factor-{kappa}B-Dependent Mechanisms in Breast Cancer Cells Regulate Tumor Burden and Osteolysis in Bone Cancer Res., April 15, 2005; 65(8): 3209 - 3217. [Abstract] [Full Text] [PDF]
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E. Corey, L. G. Brown, J. A. Kiefer, J. E. Quinn, T. E.M. Pitts, J. M. Blair, and R. L. Vessella Osteoprotegerin in Prostate Cancer Bone Metastasis Cancer Res., March 1, 2005; 65(5): 1710 - 1718. [Abstract] [Full Text] [PDF]
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P. Bhatia, M. M. Sanders, and M. F. Hansen Expression of Receptor Activator of Nuclear Factor-{kappa}B Ligand Is Inversely Correlated with Metastatic Phenotype in Breast Carcinoma Clin. Cancer Res., January 1, 2005; 11(1): 162 - 165. [Abstract] [Full Text] [PDF]
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 L. C. Hofbauer and M. Schoppet Clinical Implications of the Osteoprotegerin/RANKL/RANK System for Bone and Vascular Diseases JAMA, July 28, 2004; 292(4): 490 - 495. [Abstract] [Full Text] [PDF]
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G. L. Barnes, K. E. Hebert, M. Kamal, A. Javed, T. A. Einhorn, J. B. Lian, G. S. Stein, and L. C. Gerstenfeld Fidelity of Runx2 Activity in Breast Cancer Cells Is Required for the Generation of Metastases-Associated Osteolytic Disease Cancer Res., July 1, 2004; 64(13): 4506 - 4513. [Abstract] [Full Text] [PDF]
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E. Grimaud, L. Soubigou, S. Couillaud, P. Coipeau, A. Moreau, N. Passuti, F. Gouin, F. Redini, and D. Heymann Receptor Activator of Nuclear Factor {kappa}B Ligand (RANKL)/Osteoprotegerin (OPG) Ratio Is Increased in Severe Osteolysis Am. J. Pathol., November 1, 2003; 163(5): 2021 - 2031. [Abstract] [Full Text] [PDF]
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 H L Neville-Webbe and R E Coleman The use of zoledronic acid in the management of metastatic bone disease and hypercalcaemia Palliative Medicine, September 1, 2003; 17(6): 539 - 553. [Abstract] [PDF]
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G. L. Barnes, A. Javed, S. M. Waller, M. H. Kamal, K. E. Hebert, M. Q. Hassan, A. Bellahcene, A. J. van Wijnen, M. F. Young, J. B. Lian, et al. Osteoblast-related Transcription Factors Runx2 (Cbfa1/AML3) and MSX2 Mediate the Expression of Bone Sialoprotein in Human Metastatic Breast Cancer Cells Cancer Res., May 15, 2003; 63(10): 2631 - 2637. [Abstract] [Full Text] [PDF]
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 R. Kawaida, T. Ohtsuka, J. Okutsu, T. Takahashi, Y. Kadono, H. Oda, A. Hikita, K. Nakamura, S. Tanaka, and H. Furukawa Jun Dimerization Protein 2 (JDP2), a Member of the AP-1 Family of Transcription Factor, Mediates Osteoclast Differentiation Induced by RANKL J. Exp. Med., April 21, 2003; 197(8): 1029 - 1035. [Abstract] [Full Text] [PDF]
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 O. Sezer, U. Heider, I. Zavrski, C. A. Kuhne, and L. C. Hofbauer RANK ligand and osteoprotegerin in myeloma bone disease Blood, March 15, 2003; 101(6): 2094 - 2098. [Abstract] [Full Text] [PDF]
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 S. Barille-Nion, B. Barlogie, R. Bataille, P. L. Bergsagel, J. Epstein, R. G. Fenton, J. Jacobson, W. M. Kuehl, J. Shaughnessy, and G. Tricot Advances in Biology and Therapy of Multiple Myeloma Hematology, January 1, 2003; 2003(1): 248 - 278. [Abstract] [Full Text] [PDF]
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L Huang, Y Y Cheng, L T C Chow, M H Zheng, and S M Kumta Tumour cells produce receptor activator of NF-{kappa}B ligand (RANKL) in skeletal metastases J. Clin. Pathol., November 1, 2002; 55(11): 877 - 878. [Full Text] [PDF]
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M. S. Bendre, D. Gaddy-Kurten, T. Mon-Foote, N. S. Akel, R. A. Skinner, R. W. Nicholas, and L. J. Suva Expression of Interleukin 8 and not Parathyroid Hormone-related Protein by Human Breast Cancer Cells Correlates with Bone Metastasis in Vivo Cancer Res., October 1, 2002; 62(19): 5571 - 5579. [Abstract] [Full Text] [PDF]
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A. Ishida, N. Fujita, R. Kitazawa, and T. Tsuruo Transforming Growth Factor-beta Induces Expression of Receptor Activator of NF-kappa B Ligand in Vascular Endothelial Cells Derived from Bone J. Biol. Chem., July 12, 2002; 277(29): 26217 - 26224. [Abstract] [Full Text] [PDF]
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S.-M. Kakonen, K. S. Selander, J. M. Chirgwin, J. J. Yin, S. Burns, W. A. Rankin, B. G. Grubbs, M. Dallas, Y. Cui, and T. A. Guise Transforming Growth Factor-beta Stimulates Parathyroid Hormone-related Protein and Osteolytic Metastases via Smad and Mitogen-activated Protein Kinase Signaling Pathways J. Biol. Chem., June 28, 2002; 277(27): 24571 - 24578. [Abstract] [Full Text] [PDF]
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H.-J. Kim, M.-J. Yoon, J. Lee, J. M. Penninger, and Y.-Y. Kong Osteoprotegerin Ligand Induces beta -Casein Gene Expression through the Transcription Factor CCAAT/Enhancer-binding Protein beta J. Biol. Chem., February 8, 2002; 277(7): 5339 - 5344. [Abstract] [Full Text] [PDF]
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 M. H. C. Lam, R. J. Thomas, K. L. Loveland, S. Schilders, M. Gu, T. J. Martin, M. T. Gillespie, and D. A. Jans Nuclear Transport of Parathyroid Hormone (PTH)-Related Protein Is Dependent on Microtubules Mol. Endocrinol., February 1, 2002; 16(2): 390 - 401. [Abstract] [Full Text] [PDF]
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 V. A. Tovar Sepulveda, X. Shen, and M. Falzon Intracrine PTHrP Protects against Serum Starvation-Induced Apoptosis and Regulates the Cell Cycle in MCF-7 Breast Cancer Cells Endocrinology, February 1, 2002; 143(2): 596 - 606. [Abstract] [Full Text] [PDF]
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B. Yi, P. J. Williams, M. Niewolna, Y. Wang, and T. Yoneda Tumor-derived Platelet-derived Growth Factor-BB Plays a Critical Role in Osteosclerotic Bone Metastasis in an Animal Model of Human Breast Cancer Cancer Res., February 1, 2002; 62(3): 917 - 923. [Abstract] [Full Text] [PDF]
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N. Giuliani, R. Bataille, C. Mancini, M. Lazzaretti, and S. Barille Myeloma cells induce imbalance in the osteoprotegerin/osteoprotegerin ligand system in the human bone marrow environment Blood, December 15, 2001; 98(13): 3527 - 3533. [Abstract] [Full Text] [PDF]
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R. K. Lindemann, P. Ballschmieter, A. Nordheim, and J. Dittmer Transforming Growth Factor beta Regulates Parathyroid Hormone-related Protein Expression in MDA-MB-231 Breast Cancer Cells through a Novel Smad/Ets Synergism J. Biol. Chem., November 30, 2001; 276(49): 46661 - 46670. [Abstract] [Full Text] [PDF]
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S. Morony, C. Capparelli, I. Sarosi, D. L. Lacey, C. R. Dunstan, and P. J. Kostenuik Osteoprotegerin Inhibits Osteolysis and Decreases Skeletal Tumor Burden in Syngeneic and Nude Mouse Models of Experimental Bone Metastasis Cancer Res., June 1, 2001; 61(11): 4432 - 4436. [Abstract] [Full Text] [PDF]
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 N. J. Horwood, J. Elliott, T. J. Martin, and M. T. Gillespie IL-12 Alone and in Synergy with IL-18 Inhibits Osteoclast Formation In Vitro J. Immunol., April 15, 2001; 166(8): 4915 - 4921. [Abstract] [Full Text] [PDF]
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 G. A. Rodan and T. J. Martin Therapeutic Approaches to Bone Diseases Science, September 1, 2000; 289(5484): 1508 - 1514. [Abstract] [Full Text]
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P. Collin-Osdoby, L. Rothe, F. Anderson, M. Nelson, W. Maloney, and P. Osdoby Receptor Activator of NF-kappa B and Osteoprotegerin Expression by Human Microvascular Endothelial Cells, Regulation by Inflammatory Cytokines, and Role in Human Osteoclastogenesis J. Biol. Chem., June 1, 2001; 276(23): 20659 - 20672. [Abstract] [Full Text] [PDF]
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), MCF-7 pcDNAIneo (
) and
MCF-7 cells overexpressing PTHrP (
) tumors. Lesion number and area
was measured from long bones of fore- and hindlimbs. Values represent
the mean ± SEM, n = 5 per group.
