Full text of DOI:10.1359/jbmr.2000.15.11.2129

JOURNAL OF BONE AND MINERAL RESEARCH
Volume 15, Number 11, 2000
©
2000 American Society for Bone and Mineral Research
Parathyroid Hormone–Related Peptide Stimulates
Proliferation of Highly Tumorigenic Human
SV40-Immortalized Breast Epithelial Cells
1
2
1
3
CHRISTOPHE CATAISSON, MICHELE LIEBERHERR, MAGALI CROS, CHRISTIANE GAUVILLE,
4
5
3
1
ANNE-MARIE GRAULET, JOEL COTTON, FABIEN CALVO, MARIE-CHRISTINE de VERNEJOUL,
6
1
JOHN FOLEY, and ZHOR BOUIZAR
ABSTRACT
Parathyroid hormone–related protein (PTHrP) is the main mediator of humoral hypercalcemia of malignancy
HHM) and it is produced by many tumors, including breast cancers. Breast epithelial cells as well as breast
(
cancer tumors and cell lines have been reported as expressing PTHrP and the PTH/PTHrP receptor,
suggesting that PTHrP may act as an autocrine factor influencing proliferation or differentiation of these cell
types. We investigated PTHrP gene expression, PTH/PTHrP receptor signaling, and PTHrP-induced mito-
genesis in three immortalized human mammary epithelial cell lines that exhibit differential tumorigenicity.
The most tumorigenic cells expressed the highest levels of PTHrP messenger RNA (mRNA) and protein. We
used reverse-transcription polymerase chain reaction (RT-PCR) and immunoblotting to detect the PTH/
PTHrP receptor transcripts and proteins in all of the three cell lines. Treatment with human PTHrP(1–34)
[
hPTHrP(1–34)] and hPTH(1–34) increased intracellular cyclic adenosine monophosphate (cAMP) but not
2
؉
2؉
free Ca in the nontumorigenic line. These agonists increased both cAMP and free Ca levels in the
moderately tumorigenic line, but only increased free Ca in the highly tumorigenic line. Application of the
2
؉
1
0
11
12
3
PTH/PTHrP receptor antagonist [Asn ,Leu ,D Trp ]PTHrP(7–34) or PTHrP antibodies reduced [ H]thy-
midine incorporation in a dose-dependent fashion in the highly tumorigenic cell line but did not affect the
other lines. Thus, treatment with a PTH/PTHrP receptor antagonist reduced cell proliferation, suggesting that
PTHrP signaling mediated by the phospholipase C (PLC) pathway stimulates proliferation of a highly
tumorigenic immortalized breast epithelial cell line. (J Bone Miner Res 2000;15:2129–2139)
Key words: parathyroid hormone–related protein, parathyroid hormone/parathyroid hormone–related hor-
mone receptor, antagonist, breast cancer, reverse-transcription polymerase chain reaction
(1)
INTRODUCTION
(HHM).
The syndrome occurs when tumor-derived
PTHrP enters the circulation and stimulates the PTH/PTHrP
ARATHYROID HORMONE–related peptide (PTHrP) initially receptor in bone and kidney cells. This results in high
was identified as a product of human tumors associated concentrations of serum calcium, decreased serum phos-
with the syndrome of humoral hypercalcemia of malignancy phate, increased excretion of cyclic adenosine monophos-
P
1
Institut National de la Sant e´ et de la Recherche M e´ dicale U349, Centre Viggo Petersen, H oˆ pital Lariboisi e` re, Paris, France.
Centre National de la Recherche Scientifique UPR 1524, Jouy en Josas, France.
Laboratoire de Pharmacologie, H oˆ pital Saint Louis, Paris, France.
Laboratoire de Biologie Endocrinienne, H oˆ pital Lariboisi e` re, Paris, France.
Commissariat Energie Nucl e´ aire, Saclay, Geif sur Yvette, France.
Indiana University Medical School, Bloomington, Indiana, U.S.A.
2
3
4
5
6
2
129

2
130
CATAISSON ET AL.
TABLE 1. TUMORIGENICITY, PHENOTYPIC CHARACTERIZATION, AND CYTOGENETIC ANALYSIS OF SV40 IMMORTALIZED
HUMAN MAMMARY EPITHELIAL CELL LINES
S1T3
Nontumorigenic
S2T2
Tumorigenic
NS2T2A1 Highly
Tumorigenic
a
Nude mouse tumors
0/13
Ϫ
Ϫ
6/19 (3 months)
Ϫ
12/12 (3 weeks)
Soft agar colony formation
Estrogen receptor status
ϩ
Ϫ
b
Ϫ
c
Pan-cytokeratin
Cytokeratin 18
100% ϩϩϩ
100% ϩϩϩ
Trisomy 1q
100% ϩϩϩ
100% ϩϩϩ
Monosomy 3p,trisomy 8q
Deletions of 11q and 13
100% ϩϩϩ
100% ϩϩ
c
Cytogenetic studies
a
7
The tumorigenic potential of ST cell lines was assayed by injecting 10 cells subcutaneously into athymic nude mice.
b
c
(18)
The expression of estrogen receptor was determined on cell extracts using ESR-EIA kits.
ϩϩϩ), (ϩϩ), strong and medium staining; %, percent of cells showing positive staining.
(
phate (cAMP), and phosphate in the urine as well as bone by breast cancer cells plays a role in the local osteolytic
(1)
(15–17)
resorption uncoupled from bone formation. It is now effects of metastatic breast cancer.
Outside of the
apparent that PTHrP is expressed in many adult and devel- PTHrP’s role in metastase development within the bone
oping tissues, and that N-terminal PTHrP signaling medi- microenvironment, it is unclear whether PTHrP also may be
ated by the PTH/PTHrP receptor regulates developmental
processes including endochondral bone growth, tooth erup-
tion, and mammary gland formation.
involved in other events associated with the progression of
these tumors. In this study, we have investigated PTHrP
gene expression, PTH/PTHrP receptor signaling, and
PTHrP-induced mitogenesis in a series of immortalized
human mammary epithelial cells that exhibit differential
tumorigenicity.
(2–4)
The human PTHrP (hPTHrP) gene is a complex transcrip-
tional unit comprised of seven exons and driven by at least
(5)
three promoters. PTHrP transcripts can undergo alterna-
tive splicing at both the 5Ј and the 3Ј ends, and the splicing
at the 3Ј end ultimately leads to the generation of three
protein isoforms of 139, 141, or 173 amino acids. Within the
N terminus of PTHrP, 8 of 13 amino acid residues are
identical to the receptor binding N terminus of PTH, and
MATERIALS AND METHODS
both peptides bind to and stimulate the PTH/PTHrP receptor Cell lines and culture
(5)
to an equivalent extent. This receptor is a G-protein–
coupled seven membrane-spanning domain member of the
S1T3 and S2T2 cell lines were established from primary
PTH/secretin receptor subfamily. The binding of ligand to cultures of normal human breast epithelial cells from two
the PTH/PTHrP receptor stimulates two signal transduction individuals by immortalization with SV40-T Ag. Like
pathways in many cell types. Activation of the adenylate breast epithelial cells in primary culture, the S1T3 cells did
cyclase (AC) pathway via Gs-protein leads to the generation
of cAMP, and activation of phospholipase C (PLC) via
Gq/11-protein leads to the hydrolysis of phosphatidyl-
not grow in soft agar and did not form tumors in nude mice.
In contrast, S2T2, which also did not grow in soft agar,
produced slow-growing tumors (Ͻ8 mm in diameter 8–10
weeks after inoculation) in nude mice. The NS2T2A1 line
was derived from an S2T2 tumor that had been grown in
nude mice, reestablished in vitro, and then repassaged in
nude mice. The NS2T2A1 line produced colonies in soft
agar and tumors that grew rapidly in nude mice. The epi-
thelial nature of the cell lines was established from the
(6,7)
inositol-4,5-bisphosphate.
This reaction generates two
second messengers, inositol-1,4,5-triphosphate, which is re-
2
ϩ
sponsible for Ca mobilization from the endoplasmic re-
(8)
ticulum, and diacylglycerol, which activates protein ki-
(9)
nase C.
PTHrP production is not limited to those neoplasms as-
sociated with HHM but is produced by a variety of cancer
cell lines and tumors, including breast cancer. PTHrP im-
munoreactivity occurs in approximately 60% of primary
breast neoplasms and more than 90% of metastatic foci in
1
00% staining for pan-cytokeratin and cytokeratin 18. The
phenotypic characterization and cytogenetic studies of these
(18,19)
cell lines are summarized in Table 1.
tured in Dulbecco’s modified Eagle’s and Ham F12 media
DMEM/F12; 1/1 [vol/vol]) without calcium (ATGC,
Cells were cul-
(10,11)
bone.
Elevated levels of PTHrP messenger RNA
(
(
mRNA) are observed in primary breast cancer patients who
(12,13)
Noisy-le Grand, France) containing 10 mM HEPES, 2 mM
glutamine, 10 ␮g/ml insulin, 5 ␮M cortisol (Sigma, St.
later develop bone metastases.
In addition, some data
suggest that high concentrations of PTHrP protein or
mRNA are associated with an advanced tumor state and Quentin-Fallavier, France), 2 ng/ml epidermal growth factor
poor prognosis in patients with breast cancer or squamous (Euromedex, Souffelweyersheim, France), 50 IU/ml peni-
(13,14)
cell carcinoma.
tastases has provided evidence that the production of PTHrP (Sigma) horse serum (ATGC).
Finally, an animal model of bone me- cillin, 50 mg/ml streptomycin, and 5% Chelex-treated

EXPRESSION AND SIGNALING OF PTHrP IN BREAST CANCER CELLS
2131
PTHrP assay
ogies, Cergy-Pontoise, France) at 37°C for 1 h in buffer
containing 1mM of each deoxynucleoside triphosphate
2
Cells were plated out in 25-cm flasks and grown until
confluent. The medium was replaced with serum-free,
growth factor–free DMEM/F12, and the cells were incu-
bated for a further 48 h. Conditioned media were collected,
and PTHrP(1–84) was measured by immunoradiometric
assay (INCSTAR Corp., Stillwater, MN, U.S.A.).
(15)
(dNTP) and 50 pmol oligo(dT)
primer. Total RNA from
the TT cell line, derived from medullary thyroid carcinoma,
and the human osteosarcoma cell line SaOs-2 were used as
positive controls for the PTHrP and PTH/PTHrP receptors,
respectively. Reverse-transcription polymerase chain reac-
tions (RT-PCRs) omitting reverse transcriptase or RNA
were used as negative controls. Complementary DNA
(cDNA) was amplified using 0.5 U Thermophilus aquaticus
polymerase (Appligene, Illkirch, France) in buffer contain-
ing 25 pmol of each specific sense and antisense primers
cAMP measurement
The cells were grown in 6-well plates until confluent. The
medium was removed, and cells were incubated in serum-
free DMEM/F12 containing 1 mM 3-isobutyl-1-methyl-
xanthine (IBMX; Sigma) for 20 minutes. The medium
was removed; fresh medium containing 1 mM IBMX and
(Genosys, Cambridge, U.K.). Oligonucleotide primers were
selected using GeneWorks (Intelligenetics, Inc., Mountain
View, CA, U.S.A.). The primers used to amplify a 285-base
(23)
pair (bp) fragment of the hPTHrP gene
were (5Ј GC-
1
0
11
either hPTHrP(1–34) or an antagonist [Asn ,Leu ,D
GACGATTCTTCCTTCACC 3Ј) sense-strand specific and
5Ј AGAGTCTAACCGGCAGAGC 3Ј) antisense-strand
1
2
Trp ]PTHrP(7–34) was added, and the incubation contin-
ued for 15 minutes. Incubation was stopped by freezing the
plates at Ϫ80°C. Cells were subjected to sonication, and the
cAMP in the medium was measured by radioimmunoassay
(
specific. The primers used to amplify a 481-bp fragment of
the hPTH/PTHrP receptor gene were (5Ј TTCACAGTCT-
TCGGCTGG 3Ј) sense-strand specific and (5Ј TT-
GAAGTCCAGTGCCAGT 3Ј) antisense-strand specific.
The mRNA of the human small ribosomal protein 14 (S14)
was used as an endogenous internal control as previously
(RIA; Immunotech, Marseilles, France).
Calcium measurement
(13)
described. The number of RT-PCR cycles was optimized
for semiquantitative assessment of the product by generat-
ing saturation curves of the amounts of RT-PCR products
against the number of cycles for 0–45 cycles of amplifica-
tion (data not shown). Amplification was carried out for 23
and 25 cycles of 95°C, 30 s; 55°C, 30 s; and 72°C, 30 s for
S14 and PTHrP cDNAs, respectively, and for 32 cycles of
2
Cells were grown on rectangular glass coverslips (3 cm )
until confluent and then incubated for 24 h in serum-free
medium before use. Cells were washed three times with
Hanks’ HEPES, pH 7.4 (137 mM NaCl, 0.441 mM
KH PO , 0.885 mM MgSO 7H O, 27.7 mM glucose, 1.25
mM CaCl , and 20 mM HEPES) and loaded with 1 ␮M
2
4
4
2
2
Fura-2/AM (Calbiochem, Meudon, France) for 40 minutes
at room temperature. The glass coverslip carrying the cells
was inserted into a cuvette containing 2.5 ml Hanks’
HEPES, pH 7.4, and the cuvette was placed in a thermal-
regulated (37°C) Hitachi F2000 spectrofluorometer (Science-
tec, Les Ullis, France). Agonists and inhibitors in the vehi-
cle (5 mM acetic acid) were added directly to the cuvette,
with continuous stirring. The Fura-2 fluorescence response
9
5°C, 60 s; 55°C, 60 s; and 72°C, 60 s for PTH/PTHrP
receptor cDNA. The resulting PCR products were resolved
by electrophoresis in a 2% agarose gel. DNA was trans-
ferred to a Genescreen nylon membrane (NEN, Paris,
France) by capillary action and hybridized overnight at
4
2°C in hybridization solution containing 500,000 counts
3
2
per minute (cpm) of the respective (␣- P)deoxyadenosine
triphosphate (dATP)–labeled oligonucleotide probe. The se-
quences for the PTHrP, PTH/PTHrP receptor, and S14
probes were
2
ϩ
to intracellular calcium concentration [Ca ]i was cali-
brated from the ratio of 340/380 nm, as described by Grynk-
(20)
2ϩ
iewicz et al.
The dissociation constant for Fura-2 Ca
5
Ј GTTTCCTGAGTTAGGTATCTGCCCTCATCATC-
AGACCCAAATCGGACGG 3Ј
Ј GCATCTGGACTTGCCAGAGCGTCCCTGAGAC-
CTCGGTGTATGGTGTGGCC 3Ј
Ј CCCGGAGTTTGATGTGTAGGGCGGTGATACC-
(20)
was taken as 224 nM. The values for Rmax and Rmin were
calculated from measurements using 25 ␮M digitonin, 4
mM ethylene glycol-bis(␤-amino ethyl ether)-N,N,NЈ,NЈ-
tetracetic acid (EGTA) and enough Tris base to raise the pH
to 8.3 or higher. Each determination in Fura-2–loaded cells
was followed by a parallel experiment under the same
conditions with non–Fura-2–loaded cells. U-73122, a direct
inhibitor of PLC, and U-73433, an inactive analogue of
5
5
CAGCTCCTTGCACCTCTGG 3Ј
The autoradiography signals were analyzed with a den-
sitometric scanner (Transidyne General Corp., Roucaire,
V e´ lizy-Villacoublay, France), and the results were ex-
pressed in arbitrary units as the ratio of PTHrP or PTH/
PTHrP receptor to S14 densitometric values.
(21)
U-73122,
were purchased from TEBU (Le Perray-en-
Yvelines, France).
RNA extraction and reverse-transcription polymerase
chain reaction analysis
Cell homogenates and Western blotting
Cells were washed three times with ice-cold phosphate-
Total RNA was isolated from confluent cell cultures by a buffered saline (PBS), pH 7.4, and scraped off into ice-cold
(22)
guanidium thiocyanate-phenol/chloroform method. Total extraction buffer (20 mM Tris-HCl, pH 7.5, 0.5 mM EGTA,
RNA (1 ␮g) was reverse transcribed with 200 U Moloney 2 mM EDTA, 0.6 mM pepstatin, 0.5 mM benzamidine, 0.1
murine leukemia virus reverse transcriptase (Life Technol- mM leupeptin, 2 mM phenylmethysulfonyl fluoride, 0.125

2
132
CATAISSON ET AL.
FIG. 1. PTHrP mRNA and protein ex-
pression in human mammary epithelial
cell lines. (A) PTHrP production was
measured in growth factor–free DMEM/
F12 medium conditioned from confluent
cells cultured for 48 h. Values are
means Ϯ SEM of three experiments per-
formed in triplicate. If no error bar is
visible, it is smaller than the symbol rep-
resenting the mean. *p Ͻ 0.05 versus
S1T3 cells. (B) Southern blot analysis of
RT-PCR products showed bands at 285
bp specific for PTHrP transcripts and a
144 bp specific for S14 transcripts frag-
ment. (C) The intensities of the hybridiz-
ing signals of the PTHrP products were
quantified by densitometric scanning and
normalized against the appropriate S14
signal. Values are means Ϯ SEM (n ϭ
3). *p Ͻ 0.5 versus S1T3 cells.
mM aprotinin, and 1 mM dithiothreitol). Cell lysates were and the cells were washed three times using 1 ml cold PBS.
centrifuged for 15 minutes at 12,000g, and the supernatants Cells were then solubilized with 0.5 N NaOH, and radio-
were stored at Ϫ80°C. Protein was determined by the Brad- activity was measured with a ␥-counter. Results were ex-
ford method.
pressed as a percentage of the specific binding in the ab-
Proteins were separated by sodium dodecyl sulfate– sence of unlabeled PTHrP [hPTHrP(1–34)]. Binding data
polyacrylamide gel electrophoresis (SDS-PAGE; 8% re- were analyzed using EBDA software (Biosoft, Miltown, NJ,
solving gel) in 25 mM Tris base, pH 8.3, and 192 mM U.S.A.).
(24)
glycine and 0.1% SDS.
phoretically to polyvinylidene difluoride (PVDF) membrane
NEN) in the same buffer with 20% ethanol for 2 h at 100
They were transferred electro-
Thymidine incorporation
(
V. Nonspecific binding to PVDF was prevented by incubat-
ing the membranes in 50 mM Tris-buffered saline (TBS),
pH 7.5, containing 150 mM NaCl, 5% skimmed milk pow-
der, 2% bovine serum albumin, and 0.1% Tween 20 for 1 h
at room temperature. The membranes were incubated over-
night at 4°C in TBA with 5 ␮g/ml polyclonal antibody
against PTH/PTHrP receptor (Biovalley, Conches, France).
The antibodies bound to the proteins on the PVDF were
detected using peroxidase-conjugated goat anti-rabbit im-
munoglobulin G (IgG; diluted 1/12,000 in TBS). The anti-
gen was detected by enhanced chemiluminescence. Molec-
ular size standards were used to estimate the apparent
molecular mass of the PTH/PTHrP receptor: myosin, 218
kDa; ␤-galactosidase, 125 kDa; bovine serum albumin, 78
kDa; and carbonic anhydrase, 44 kDa.
Cells were seeded in the wells of a 24-well plate at 30,000
cells per well and were incubated for 24 h. The medium
was then removed, and the cells were incubated in fresh
serum-free, growth factor–free DMEM/F12 containing
1
0
11
12
[
Asn ,Leu ,D Trp ]PTHrP(7–34) NH in 5 mM acetic
2
acid plus aprotinin, rabbit antibody against hPTHrP(1–34)
Peninsula Laboratories, Belmont, CA, U.S.A.), hPTHrP(1–
4), hPTH(1–34), di(Bt)cAMP, and vehicle or nonimmune
(
3
normal rabbit serum for 48 h. Fresh serum-free medium
3
containing 2 ␮Ci of [ H]thymidine (NEN) was then added
to each well for an additional 4 h. The medium was re-
moved, and the cells were washed twice with cold PBS,
once with absolute methanol, and twice with cold 5% tri-
chloroacetic acid. The cells were then solubilized in 0.5 ml
3
0
.5N NaOH, and the incorporation of [ H]thymidine was
evaluated by scintillation counting.
1
25
I-PTHrP binding
1
25
I chicken-PTHrP(1–34) binding was performed as de-
Peptides
(25,26)
scribed
for 4 h at 16°C in 250 ␮l binding buffer (100
mM NaCl, 50 mM Tris-HCl, 5mM KCl, 2 mM CaCl , and
Synthetic hPTHrP(1–34) was purchased from Bachem
2
5
125
5
% horse serum) and 1 ϫ 10 cpm
I PTHrP(1–34). (Voisins-le-Bretonneux, France); di(Bt)cAMP and hPTH(1–
1
0
11
12
Unlabeled hPTHrP at concentrations ranging from 1 ϫ 34) were purchased from Sigma. [Asn ,Leu ,D Trp ]-
Ϫ10
Ϫ6
1
0
to 1 ϫ 10 M was used to assess displacement. At PTHrP(7–34) was synthesized using a fully automatic pep-
the end of the incubation, an aliquot of medium was re- tide synthesizer (model 431A; Applied Biosystems, Perkin
moved for counting. The remaining medium was aspirated Elmer, Les Ullis, France).

EXPRESSION AND SIGNALING OF PTHrP IN BREAST CANCER CELLS
2133
FIG. 3. PTH/PTHrP receptor protein expression in human
mammary epithelial cell lines. Fifty micrograms of cell
homogenates were separated by electrophoresis on SDS-
PAGE, transferred to PVDF membranes, and probed with
antibodies against PTH/PTHrP receptor. A predominant
band at 78 kDa of similar intensity was observed in extracts
from all three lines.
cultures of each cell type. NS2T2A1-conditioned medium
contained the highest concentrations of PTHrP and the
S2T2-conditioned media contained intermediate amounts.
The S1T3-conditioned media contained relatively low con-
centrations of immunodetectable peptide (Fig. 1A). Next,
we determined whether the mRNA concentration in the
cells was consistent with the iPTHrP levels in the condi-
tioned media. Using semiquantitative RT-PCR analysis, we
found that NS2T2A1 cells expressed the highest levels,
S2T2 cells expressed intermediate levels, and S1T3 cells
expressed the lowest levels of PTHrP mRNA, approxi-
mately one-third of that expressed by the NS2T2A1 line
FIG. 2. Semiquantitative RT-PCR analysis of PTH/
PTHrP receptor mRNA in human mammary epithelial cell
lines. (A) Southern blot analysis of RT-PCR products
showed bands at 481 bp for the PTH/PTHrP receptor tran-
scripts and at 144 bp specific for S14 transcripts. (B) The
intensities of the hybridizing signals of the PCR products
were quantified with densitometric scanning and normalized
against the appropriate S14 signal. Values are means Ϯ
SEM (n ϭ 3). *p Ͻ 0.5 versus S1T3 cells.
(Figs. 1B and 1C). The relative levels of PTHrP mRNA
expression observed in the three cell lines reflected the
PTHrP protein levels measured in conditioned media for
Statistical methods
Data were analyzed by analysis of variance (ANOVA) each cell type. Among these three cell lines, higher levels of
using Fisher’s projected least significant difference (PLSD) PTHrP gene expression were associated with increased tu-
or Scheff e´ ’s F test to compare means. The value of p Ͻ 0.05 morigenicity.
was significant.
Expression of the PTH/PTHrP receptor
RESULTS
Next, we investigated PTH/PTHrP receptor expression in
the three cell lines. Using semiquantitative RT-PCR analy-
sis, we evaluated PTH/PTHrP receptor mRNA levels in the
PTHrP expression of breast epithelial cell lines
In the following studies, we have employed three cell three lines. As shown in Fig. 2A, the amplified PTH/PTHrP
lines (S1T3, S2T2, and NS2T2A1) derived from SV40 large receptor bands from NS2T2A1 and S1T3 cells were of
T-antigen immortalized breast epithelial cells. In tumorige- similar intensity, whereas the band representing PTH/
nicity assays using nude mice, S1T3 cells did not produce PTHrP receptor mRNA from S2T2 cells appeared to be
tumors, S2T2 cells produced small slow-growing tumors, approximately 1/4 the intensity of the other two lines (Fig.
and the third line NS2T2A1 produced aggressive tumors. 2B). We also used Western blotting to detect PTH/PTHrP
The characteristics of these cells and the chromosomal receptor protein expression in extracts from each of the
abnormalities they contain suggested that they could pro- lines. As shown in Fig. 3, a major immunoreactive band of
vide an in vitro model of breast tumor progression (Table similar intensity was observed at ϳ78 kDa in all three lines.
(18,19)
1
).
As a first step in characterizing PTHrP gene ex- In addition, we performed receptor binding assays on intact
pression in these lines, we measured immunoreactive cells to evaluate relative number of plasma membrane
PTHrP (iPTHrP) levels in conditioned media from confluent PTHrP binding sites for each cell type. Figure 4 shows that

2
134
CATAISSON ET AL.
FIG. 5. Effects of PTH(1–34) and PTHrP(1–34) on
cAMP accumulation in S1T3, S2T2, and NS2T2A1 cell
lines. Cells were incubated for 15 minutes at 37°C in
medium containing 1 mM IBMX, the substance to be tested,
or its vehicle (control). Values are the means Ϯ SEM (n ϭ
1
25
FIG. 4.
I-labeled PTHrP binding to human mammary
epithelial cell lines. This figure represents competition of
1
25
I-labeled PTHrP to S1T3 (open squares), S2T2 (circles),
1
2). a, p Ͻ 0.001; b, p Ͻ 0.01 significantly different from
and NS2T2A1 (triangles) cells by various doses of unla-
beled PTHrP. Specific binding was calculated by subtract-
ing nonspecific binding (NSB ϭ counts per minute bound in
the control values.
the presence of 1 ␮M PTHrP) from total binding (B ϭ
0
3
3
4) and hPTH(1–34). As shown in Fig. 5, both hPTHrP(1–
4) and hPTH(1–34) produced a dose-dependent increase in
counts per min bound in the absence of unlabeled PTHrP).
Values are shown as a percentage of the specific binding in
the absence of unlabeled PTHrP. Values are the mean Ϯ
SEM of duplicates and are representative of three separate
experiments. If no error bar is visible, it was smaller than
the symbol representing the mean.
cAMP levels in the S1T3 cells and one of a smaller mag-
nitude in the S2T2 cell line. In contrast, hPTHrP(1–34),
even at high concentration (1 ␮M), had no effect on the
cAMP response in NS2T2A1 cells, whereas forskolin in-
duced a 6-fold increase in cAMP production in all the lines
(
Fig. 5). To evaluate activation of the PLC pathway, we
2
ϩ
monitored intracellular [Ca ]i levels. Both hPTHrP(1–34)
specific binding for I PTHrP ligand was detected for each and hPTH(1–34) caused a dose-dependent increase in
cell line. Schatchard analysis of these competitive displace- [Ca ]i in NS2T2A1 cells (Table 2). The amplitudes of the
ment experiments revealed the presence of 364, 268, and [Ca ]i responses to the two agonists were similar. The
02 fmol/mg protein of a single class of binding sites, with increase in [Ca ]i was transient, with a maximum at 10 s.
1
25
2
ϩ
2
ϩ
2
ϩ
3
an affinity of ϳ52, 55, and 27 nM, respectively, for S1T3, The peak was followed by a plateau that was above the
S2T2, and NS2T2A1 cells. The results from the receptor basal calcium concentration (19 Ϯ 3%; n ϭ 12; Fig. 6A).
2
ϩ
binding assays suggested that all three lines contained a Neither hPTHrP nor hPTH had any effect on the [Ca ]i in
similar number of PTHrP binding sites on the plasma mem- S1T3 cells (data not shown), whereas they caused a small
brane. At this point, it is unclear as to why there was a increase in S2T2 cells (Table 2). Together, these results
discrepancy between the RT-PCR and immunoblotting indicated that both N-terminal PTHrP and PTH were capa-
analysis of PTH/PTHrP receptor expression observed in ble of inducing second messenger cascades in each cell
S2T2 line, but this might reflect posttranscriptional regula- type; however, it appeared that the PLC pathway is exclu-
tion of this mRNA. Together, these results suggested that all sively induced in the highly tumorigenic NS2T2A1 cells,
three cell lines express PTH/PTHrP receptor mRNA and whereas the AC pathway is selectively induced in the non-
protein and specific PTHrP binding observed with these tumorigenic S1T3 cells.
cells.
We used pharmacologic tools to investigate the relative
2
ϩ
contributions of extracellular Ca influx and intracellular
2
ϩ
2ϩ
Ca release to the increased intracellular [Ca ]i response
observed when NS2T2A1 and S2T2 lines were treated with
Signaling pathways of PTHrP
Once we had established that the lines expressed PTHrP hPTHrP(1–34) and hPTH(1–34). EGTA, a chelator of ex-
2
ϩ
and a plasma membrane PTH/PTHrP receptor, we wanted to tracellular Ca , reduced (50 Ϯ 10%;n ϭ 4) the transient
determine whether N-terminal PTHrP or PTH triggered a peak and abolished the sustained plateau phase of this
similar set of second messenger cascades in each cell type. response (Fig. 6B). Nifedipine (1 ␮M) and verapamil (1
To evaluate activation of the AC pathway, we monitored ␮M), L-type calcium channel blockers, also decreased the
intracellular cAMP levels in cells treated with hPTHrP(1– transient peak and abolished the plateau phase of this re-

EXPRESSION AND SIGNALING OF PTHrP IN BREAST CANCER CELLS
2135
TABLE 2. DOSE-DEPENDENT EFFECTS OF hPTHrP(1-34) AND hPTH(1-34) ON INTRACELLULAR
2
ϩ
Ca CONCENTRATION IN NS2T2A1 AND S2T2 CELLS
2
ϩ
⌬
[Ca ]i nM
NS2T2A1 cells
hPTHrP(1-34)
S2T2 cells
Concentration
pM
hPTH(1-34)
hPTHrP(1-34)
hPTH(1-34)
1
9.1 Ϯ 1.3
10.3 Ϯ 1.1
7.0 Ϯ 2.0
6.6 Ϯ 1.5
5.0 Ϯ 3.0
13.6 Ϯ 3.2
35.0 Ϯ 4.1*
1
0 pM
00 pM
nM
0 nM
00 nM
29.1 Ϯ 3.1*
34.6 Ϯ 3.5*
†
†
1
71.6 Ϯ 3.9
70.9 Ϯ 3.1
35.6 Ϯ 3.7*
†
†
†
†
1
116.3 Ϯ 6.6
115.7 Ϯ 6.1
53.6 Ϯ 2.1
53.6 Ϯ 3.2
†
†
†
†
1
199.4 Ϯ 3.9
184.9 Ϯ 7.9
66.6 Ϯ 3.2
61.6 Ϯ 1.1
†
†
†
†
1
256.5 Ϯ 6.9
224.5 Ϯ 7.1
75.6 Ϯ 2.8
70.3 Ϯ 3.1
2
ϩ
2ϩ
[
Ca ]i was measured at t ϭ 10 s after adding hPTHrP or hPTH to Fura-2 loaded cells. Basal [Ca ]i in NS2T2A1 was 105.6 Ϯ 8
2
ϩ
2ϩ
nM (n ϭ 12) and basal [Ca ]i in S2T2 was 110 Ϯ 4 nM (n ϭ 12). The vehicle (5 mM acetic acid) did not affect [Ca ]i. Results
2
ϩ
are expressed as ⌬[Ca ]i nM for the increase induced by both agonists versus the corresponding basal level. Values are means Ϯ SEM
n ϭ 4).
p Ͻ 0.01.
(
*
†
p Ͻ 0.001 versus controls.
sponse (Fig. 6C, data shown for nifedipine only). Three eration, we employed hPTHrP(1–34) neutralizing antibod-
3
micromolar U-73122, a direct inhibitor of PLC but not the ies. Treatment with anti-hPTHrP antibody decreased [ H]thy-
inactive analog of U-73343, abolished the transient peak but midine incorporation in the NS2T2A1 highly tumorigenic line
3
had no effect on the plateau phase of this response (Fig. (Table 3). The [ H]thymidine incorporation decreased as the
6
D). Identical experiments on S2T2 cells produced similar antibody concentration increased (from 1:800 dilution to 1:100
results (data not shown). Thus, N-terminal PTHrP and PTH dilution). The same neutralizing antibody had no effect on
2
ϩ
3
increased the intracellular free Ca
concentration in [ H]thymidine incorporation into S1T3 or S2T2 cells (Table
NS2T2A1 and S2T2 cells via two mechanisms, one involv- 3). Taken together, these results suggest that endogenously
2
ϩ
ing Ca influx through voltage-gated L-type calcium chan- produced PTHrP stimulates the proliferation of cultured
2
ϩ
nels and the other by mobilizing Ca from the endoplasmic NS2T2A1 cells but not S1T3 or S2T2 cells.
reticulum.
We next investigated whether the PTH/PTHrP receptor
mediated PTHrP-induced proliferation in NS2T2A1 cells by
employing a specific PTH/PTHrP receptor antagonist
Stimulation of the proliferation of NS2T2A1 cells by
endogenous PTHrP
1
0
11
12
[Asn ,Leu ,D Trp ]PTHrP(7–34). The exchange of
amino acids in position 10 and 11 of PTHrP(7–34) with
In the next set of experiments, we wanted to determine if position 10 and 11 of PTH(7–34) along with the replace-
PTHrP signaling influenced proliferation of the cells. To ment of Glu12 with D-Trp results in a very potent antago-
(27)
this end, we added hPTHrP(1–34), hPTH(1–34), and di(Bt)- nist devoid of detectable agonism.
Cells were incubated
cAMP in growth factor–free media to cultures and then with this PTH/PTHrP receptor antagonist and this resulted
3
3
monitored DNA synthesis by evaluating [ H]thymidine in- in dose-dependent decreases of [ H]thymidine incorpora-
corporation at 48 h. As shown in Fig. 7, exogenous tion in NS2T2A1 cells, whereas S1T3 and S2T2 cells were
hPTHrP(1–34) and hPTH(1–34) did not significantly alter unaffected by this agent (Fig. 8). The maximal effect was
thymidine incorporation rates in S1T3, S2T2, or NS2T2A1. obtained with 1 ␮M antagonist. This concentration was
In contrast, the addition of di(Bt)cAMP to each cell type used to establish a time course of the antagonist’s effect on
3
inhibited DNA synthesis in all cell lines with a minimal [ H]thymidine incorporation in NS2T2A1 cells. Decreases
Ϫ4
3
effective dose of 10 M. Under these assay conditions, in [ H]thymidine incorporation were observed only after
exogenously added N-terminal PTHrP did not appear to 48 h incubation with the antagonist, and the magnitude of
stimulate proliferation of S1T3 and S2T2 cells, whereas this effect on proliferation was still 60% after 72 h incuba-
stimulation of the AC arm of the PTH/PTHrP receptor tion (17,044 Ϯ 2065 cpm vs. 41,097 Ϯ 3065 cpm for
signaling pathway may inhibit proliferation.
control; p Ͻ 0.05). Thus, it appears that a portion of the
Ϫ7
Although the addition of hPTHrP(1–34) at 10 M failed proliferative effect of endogenous PTHrP on NS2T2A1
to increase significantly proliferation of the NS2T2A1 cells, cells can be inhibited by PTH/PTHrP receptor antagonists.
individual replicates of this experiment showed a trend of
3
increased [ H]thymidine incorporation. Based on these ob-
servations, we speculated that high levels of endogenous
DISCUSSION
PTHrP in these cells might minimize the response to exog-
enous PTHrP over the 48-h time course of these assays. To
address the influence of endogenous PTHrP on cell prolif- mammary physiology,
PTHrP plays a central role in breast development and
(4,28)
and several lines of evidence

2
136
CATAISSON ET AL.
pects of the disease that precede metastasis to bone. In this
study we have employed a series of SV40 immortalized
human mammary epithelial cells that exhibit differential
tumorigenicity as a model system to evaluate PTHrP and
PTH/PTHrP gene expression, N-terminal PTHrP signaling,
and the influence of the peptide on proliferation in vitro.
Previous surveys have reported that several breast cancer
cell lines express both PTHrP and PTH/PTHrP receptor
mRNA, but there are a subset of lines that do not express
(31)
mRNAs for the ligand or the receptor.
By evaluating
PTHrP protein and mRNA expression in the SV40 immor-
talized human mammary epithelial cell lines, we found that
PTHrP protein and mRNA expression levels of the various
lines are associated with tumorigenicity. The highly tumor-
igenic NS2T2A1 and tumorigenic S2T2 cells expressed
significantly greater amounts of PTHrP protein and mRNA
than the nontumorigenic S1T3 cells. RT-PCR amplified
specific PTH/PTHrP receptor transcripts in all of the cells.
Results from Western blots and receptor binding assays
suggested that overall PTH/PTHrP receptor protein levels as
well as PTHrP binding sites on the plasma membrane were
similar for all three lines. It is possible that this series of
immortalized breast epithelial cells with differential tumor-
igenicity might prove to be a useful model to study PTHrP
and PTH/PTHrP receptor gene expression.
In many cell types, the binding of ligand to the PTH/
PTHrP receptor stimulates both the AC and the PLC second
messenger pathway. At this point, PTH/PTHrP receptor
signaling has not been studied comprehensively in breast
cancer cells. Previous studies employing MCF-7 cells have
reported that PTHrP(1–34) stimulated increases in cAMP,
(31)
but effects on the PLC signaling arm were not evaluated.
Using cAMP assays and Fura-2 fluorescence to evaluate
intracellular calcium levels, we evaluated the response of
FIG. 6. Effects of PTH(1–34) and PTHrP(1–34) on the S1T3, S2T2, and NS2T2A1 cells to hPTHrP(1–34) and
2
ϩ
intracellular Ca
concentration in NS2T2A1 cells. (A) hPTH(1–34). We found no evidence that N-terminal PTHrP
Direct effect of 10 nM PTHrP or PTH. (B) Cells were or PTH stimulation increased cAMP levels of the highly
treated for 30 s with 2 mM EGTA before adding 10 nM tumorigenic cell line, NS2T2A1; however, forskolin treat-
PTHrP or PTH. (C) Cells were treated for 1 minute with 1 ment triggered a robust cAMP response indicating that AC
␮
M nifedipine before adding 10 nM PTHrP or PTH. (D) was not defective in this line. Both peptides increased the
Cells were incubated for 1 minute with 1 ␮M U-73122, a intracellular cAMP levels in tumorigenic S2T2 and nontu-
direct inhibitor of PLC, before adding 10 nM PTHrP or morigenic S1T3 cells. In contrast, N-terminal PTHrP and
PTH. These results are representative of at least four repli- PTH stimulated a rise in intracellular calcium that can be
cates for each experiment.
attributed in part to activation of the PLC pathway in the
transformed NS2T2A1 cell line and to a lesser extent in the
S2T2, but it did not stimulate this pathway in S1T3 cells.
Thus, N-terminal PTHrP and PTH signaling is mediated
exclusively through the PLC pathway in the most tumori-
genic cell type, NS2T2A1.
suggest that this peptide may be an important factor in the
progression of diseases of breast tissue. A recent survey of
invasive breast carcinomas indicated that 100% expressed
PTHrP immunoreactivity and 96% were labeled with PTH/
There are several potential explanations for the selective
Breast cancer tumors and activation of a single transduction pathway by N-terminal
(29)
PTHrP receptor antibodies.
metastases that express high levels of PTH/PTHrP receptor PTHrP and PTH in the S1T3 and NS2T2A1 cells. Several
mRNA and protein have a significantly increased number of studies have found that that the cell surface density of the
cells that label with antibodies to proliferation marker Ki- PTH/PTHrP receptor influenced ligand induced activation
6
7, suggesting that this receptor may be associated with an of the PLC pathway. In kidney cell lines transfected with
(30)
increased proliferation rate in these tumors.
There are PTH/PTHrP receptor expression vectors, activation of the
elevated amounts of PTHrP mRNA in primary breast cancer PLC pathway appears to be dependent on higher densities of
(12,13)
(32)
patients who later develop bone metastases.
Together, cell surface receptors.
The S1T3 and NS2T2A1 cells
these findings suggest that the peptide could influence as- appear to express comparable levels of PTH/PTHrP recep-

EXPRESSION AND SIGNALING OF PTHrP IN BREAST CANCER CELLS
2137
FIG.
7. Effect
of
di(Bt)cAMP, PTH(1–34),
and PTHrP(1–34) on the
growth of human mammary
epithelial cell lines. All cells
were exposed to the various
agonist for 48 h in media
without serum and growth
factors. (A) S1T3 cells;
(B) S2T2 cells; and (C)
NS2T2A1 cells. Values are
means Ϯ SEM (n ϭ 3).
*
p Ͻ 0.05 versus control.
TABLE 3. NEUTRALIZING ANTIBODY AGAINST PTHrP(1-34)
DECREASES IN THE GROWTH OF NS2T2A1 CELLS
There is emerging evidence that PTHrP stimulates pro-
liferation of a subset of tumor cell lines, suggesting that the
peptide may play a role as an autocrine factor in the devel-
opment of some neoplasms. Neutralizing antibodies have
been used to abolish the mitogenic effect of exogenous
3
[
H]thymidine incorporation (cpm/well)
PTHrP
(36–38)
antibody
S1T3 cells
10,700 Ϯ 320
S2T2 cells
NS2T2A1 cells
PTHrP(1–34) on cancer cells.
Other studies have used
antisense technology to inhibit the production of PTHrP and
0
8,640 Ϯ 340
24,237 Ϯ 1848
(39,40)
to reduce tumor development in vivo.
In Walker car-
0
0
0
1
.125% 10,940 Ϯ 940
9,070 Ϯ 1010 22,018 Ϯ 3798
cinoma cells, a mammary gland–derived cell type used in
animal models of HHM, N-terminal PTHrP and PTH ex-
clusively signal through the PLC pathway, and treatment
with these agonists stimulates proliferation of these cells in
.25%
.5%
%
12,000 Ϯ 450
11,000 Ϯ 670
10,720 Ϯ 1050 8,370 Ϯ 740
9,360 Ϯ 810
7,780 Ϯ 400
15,703 Ϯ 1537*
3,646 Ϯ 295*
2,326 Ϯ 50*
(38)
All cells were exposed to antibody (shown as a percentage of the vitro. Addition of N-terminal PTHrP and PTH to MCF-7,
concentration, [vol/vol]) for 48 h in media without serum and a breast cancer line that expresses low levels of PTHrP,
3
(31)
growth factors. Values are means Ϯ SEM (n ϭ 4).
p Ͻ 0.05 versus control (nonimmune rabbit serum).
increased [ H]thymidine incorporation.
Addition of
*
N-terminal PTHrP and PTH to S1T3, S2T2, and NS2T2A1
cells did not appear to affect proliferation, whereas treat-
3
ment of each of the lines with di(Bt)cAMP reduced [ H]thy-
midine incorporation. These findings lead to speculation
that stimulation of the AC arm to the PTH/PTHrP receptor
signaling pathway may inhibit proliferation of these cell
types, as has been previously reported for Walker carcinoma
cells. We found that incubation with a neutralizing antibody
tor protein and have similar numbers of plasma membrane
PTHrP binding sites; therefore, it is possible that other
factors account for the distinct signal transduction responses
to N-terminal PTHrP and PTH observed in these two cell
lines. Changes in signaling properties also could result from
the expression of various subpopulations of receptors. Ma-
lignant squamous carcinoma cells appear to have an alter-
native PTH/PTHrP receptor that responds to N-terminal
PTHrP or PTH by stimulating a rise in intracellular calcium
3
against PTHrP substantially decreases [ H]thymidine incor-
poration in the highly tumorigenic cell line NS2T2A1 but
had no effect on S1T3 or S2T2 cells. Incubation of
NS2T2A1 cells with the PTH/PTHrP receptor antagonist
10
11
12
3
[
Asn ,Leu ,D Trp ]PTHrP(7–34) also decreased [ H]thy-
(33,34)
but not cAMP.
Our findings do not exclude the pos-
midine incorporation in NS2T2A1. Together, these findings
are consistent with the concept that endogenously produced
PTHrP stimulates proliferation in NS2T2A1 cells and that
the PTH/PTHrP receptor is the mediator of at least a portion
of the PTHrP-induced mitogenesis. In the context of the
results from the N-terminal PTHrP and PTH signal trans-
sibility that NS2T2A1 and S2T2 cells may express a similar
PLC/calcium-coupled receptor along with the “classical”
PTH/PTHrP receptor. Finally, the transformation of the
cells also could interfere with the molecular mechanisms
that mediate the specificity of PTH/PTHrP receptor signal-
ing. It has been proposed that the cell-type specific distri- duction studies, these findings imply that the mitogenic
bution or compartmentalization of G-protein subunits and effect of PTHrP in the highly tumorigenic NS2T2A1 cells
2
ϩ
their associated signal transduction effectors could govern was mediated by PLC/Ca signaling. Thus, our findings
(35)
the differential activity of the PTH/PTHrP receptor.
expect that S1T3, S2T2, and NS2T2A1 cells will be useful PTHrP receptor and selective activation of the PLC/Ca
reagents to study the relationship between the PTHrP signal signal transduction pathway leads to proliferation and pro-
transduction and tumorigenicity. gression of a subset of breast cancers.
We raise the possibility that PTHrP stimulation of the PTH/
2
ϩ

2
138
CATAISSON ET AL.
4
. Wysolmerski JJ, Stewart AF 1998 The physiology of parathy-
roid hormone-related protein: An emerging role as a develop-
mental factor. Annu Rev Physiol 60:431–460.
5
. Broadus AE, Stewart AF 1994 Parathyroid Hormone-Related
Protein Structure, Processing and Physiological Actions. The
Parathyroids. Raven Press, New York, NY, U.S.A., pp. 259–
2
94.
6
7
. Abou-Samra A, Juppner H, Force T, Freeman MW, Kong XF,
Schipani E, Urena P, Richards J, Bonventre JV, Potts JJ,
Kronenberg HM, Segre GV 1992 Expression cloning of a
common receptor for parathyroid hormone and parathyroid
hormone-related peptide from rat osteoblast-like cells: A sin-
gle receptor stimulates intracellular accumulation of both
cAMP and inositol trisphosphates and increases intracellular
free calcium. Proc Natl Acad Sci USA 89:2732–2736.
. Dunlay R, Hruska K 1990 PTH receptor coupling to phospho-
lipase C is an alternate pathway of signal transduction in bone
and kidney. Am J Physiol 258:F223–F231.
8
. Berridge MJ 1993 Inositol trisphosphate and calcium signal-
ling. Nature 361:315–325.
9
. Nishizuka Y 1995 Protein kinase C and lipid signaling for
sustained cellular responses. FASEB J 9:484–496.
1
0. Powell GJ, Southby J, Danks JA, Stillwell RG, Hayman JA,
Henderson MA, Bennett RC, Martin TJ 1991 Localization of
parathyroid hormone-related protein in breast cancer metasta-
ses: Increased incidence in bone compared with other sites.
Cancer Res 51:3059–3061.
1
1. Southby J, Kissin MW, Danks JA, Hayman JA, Moseley JM,
Henderson MA, Bennett RC, Martin TJ 1990 Immunohisto-
chemical localization of parathyroid hormone-related protein
in human breast cancer. Cancer Res 50:7710–7716.
1
2. Bouizar Z, Spyratos F, Deytieux S, de Vernejoul MC, Julli-
enne A 1993 Polymerase chain reaction analysis of parathy-
roid hormone-related protein gene expression in breast cancer
patients and occurrence of bone metastases. Cancer Res 53:
5
076–5078.
1
3. Bouizar Z, Spyratos F, de Vernejoul MC 1999 The parathyroid
hormone-related protein (PTHrP) gene: Use of downstream
TATA promotor and PTHrP 1–139 coding pathways in pri-
mary breast cancers vary with the occurrence of bone metas-
tasis. J Bone Miner Res 14:406–414.
1
0
11
12
FIG. 8. Effect of [Asn ,Leu ,D Trp ]PTHrP(7–34) on
the growth of human mammary epithelial cell lines. All 14. Pecherstorfer M, Schilling T, Blind E, Zimmer RI, Baumgart-
cells were exposed to the antagonist for 48 h in media
without serum and growth factors (A) S1T3 cells; (B) S2T2
cells; and (C) NS2T2A1 cells. Values are means Ϯ SEM
ner G, Ziegler R, Raue F 1994 Parathyroid hormone-related
protein and life expectancy in hypercalcemic cancer patients.
J Clin Endocrinol Metab 78:1268–1270.
1
1
5. Guise TA, Yin JJ, Taylor SD, Kumagai Y, Dallas M, Boyce
BF, Yoneda T, Mundy GR 1996 Evidence for a causal role of
parathyroid hormone-related protein in the pathogenesis of
human breast cancer-mediated osteolysis. J Clin Invest 98:
(n ϭ 4). *p Ͻ 0.05 versus control.
1
544–1549.
ACKNOWLEDGMENTS
This work was supported by grants 6228 and 9689 from
6. Yin JJ, Selander K, Chirgwin JM, Dallas M, Grubbs BG,
Wieser R, Massague J, Mundy GR, Guise TA 1999 TGF-beta
signaling blockade inhibits PTHrP secretion by breast cancer
cells and bone metastases development. J Clin Invest 103:197–
the Association pour la Recherche contre le Cancer (ARC).
2
06.
1
1
7. Thomas RJ, Guise TA, Yin JJ, Elliott J, Horwood NJ, Martin
TJ, Gillespie MT 1999 Breast cancer cells interact with osteo-
blasts to support osteoclast formation. Endocrinology 140:
REFERENCES
1
2
3
. Broadus AE, Mangin M, Ikeda K, Insogna KL, Weir EC,
Burtis WJ, Stewart AF 1988 Humoral hypercalcemia of can-
cer. Identification of a novel parathyroid hormone-like peptide.
N Engl J Med 319:556–563.
. Karaplis AC, Luz A, Glowacki J, Bronson RT, Tybulewicz
VL, Kronenberg HM, Mulligan RC 1994 Lethal skeletal dys-
4
451–4458.
8. Berthon P, Goubin G, Dutrillaux B, Degeorges A, Faille A,
Gespach C, Calvo F 1992 Single-steep transformation of hu-
man breast epithelial cells by SV40 large T oncogene. Int J
Cancer 52:92–97.
plasia from targeted disruption of the parathyroid hormone- 19. Lebeau J, Gerbault-Seureau M, Lemieux N, Apiou F, Calvo F,
related peptide gene. Genes Dev 8:277–289.
Berthon P, Goubin G, Dutrillaux B 1995 Loss of chromosome
3p arm differentiating tumorigenic from non-tumorigenic cells
derived from the same SV40-transformed human mammary
epithelial cells. Int J Cancer 60:244–248.
. Philbrick WM, Dreyer BE, Nakchbandi IA, Karaplis AC 1998
Parathyroid hormone-related protein is required for tooth erup-
tion. Proc Natl Acad Sci USA 95:11846–11851.

EXPRESSION AND SIGNALING OF PTHrP IN BREAST CANCER CELLS
2139
2
0. Grynkiewicz G, Poenie M, Tsien RY 1985 A new generation
tide receptor density modulates activation of phospholipase C
and phosphate transport by PTH in LLC-PK1 cells. Endocri-
nology 136:3884–3891.
2
ϩ
of Ca indicators with greatly improved fluorescence prop-
erties. J Biol Chem 260:3440–3450.
2
1. Bleasdale JE, Bundy GL, Bunting S, Fitzpatrick FA, Huff RM, 33. Orloff JJ, Ganz MB, Ribaudo AE, Burtis WJ, Reiss M, Mil-
Sun FF, Pike JE 1989 Inhibition of phospholipase C dependent
processes by U-73, 122. Adv Prostaglandin Thromboxane
Leukot Res 19:590–593.
stone LM, Stewart AF 1992 Analysis of PTHRP binding and
signal transduction mechanisms in benign and malignant squa-
mous cells. Am J Physiol 262:E599–E607.
2
2. Chomczynski P, Sacchi N 1987 Single step method of RNA 34. Orloff JJ, Kats Y, Urena P, Schipani E, Vasavada RC,
isolation by acid guanidium thiocianate phenol chloroform
extraction. Anal Biochem 162:321–329.
3. Mangin M, Webb AC, Dreyer BE, Posillico JT, Ikeda K, Weir
EC, Stewart AF, Bander NH, Milstone L, Barton DE, Francke
U, Broadus AE 1988 Identification of a cDNA encoding a
Philbrick WM, Behal A, Abou Samra A, Segre GV, Juppner H
1995 Further evidence for a novel receptor for amino-terminal
parathyroid hormone-related protein on keratinocytes and
squamous carcinoma cell lines. Endocrinology 136:3016–
3023.
2
parathyroid hormone-like peptide from a human tumor asso- 35. Maeda S, Wu S, Juppner H, Green J, Aragay AM, Fagin JA,
ciated with humoral hypercalcemia of malignancy. Proc Natl
Acad Sci USA 85:597–601.
4. Laemmli UK 1970 Cleavage of structural proteins during the
assembly of the head of bacteriophage T4. Nature 227:680–
Clemens TL 1996 Cell-specific signal transduction of parathy-
roid hormone (PTH)-related protein through stably expressed
recombinant PTH/PTHrP receptors in vascular smooth muscle
cells. Endocrinology 137:3154–3162.
2
2
6
85.
36. Burton PB, Knight DE 1992 Parathyroid hormone-related pep-
tide can regulate the growth of human lung cancer cells, and
may form part of an autocrine TGF-alpha loop. FEBS Lett
305:228–232.
37. Iwamura M, Abrahamsson PA, Foss KA, Wu G, Cockett AT,
Deftos LJ 1994 Parathyroid hormone-related protein: A poten-
tial autocrine growth regulator in human prostate cancer cell
lines. Urology 43:675–679.
5. Gaich G, Orloff JJ, Atillasoy EJ, Burtis WJ, Ganz MB, Stewart
AF 1993 Amino-terminal parathyroid hormone-related pro-
tein: Specific binding and cytosolic calcium responses in rat
insulinoma cells. Endocrinology 132:1402–1409.
2
6. Jobert AS, Leroy C, Butlen D, Silve C 1997 Parathyroid
hormone-induced calcium release from intracellular stores in a
human kidney cell line in the absence of stimulation of cyclic
adenosine 3Ј,5Ј-monophosphate production. Endocrinology 38. Benitez VJ, Esbrit P 1994 Proliferative effect of parathyroid
1
38:5282–5292.
hormone-related protein on the hypercalcemic Walker 256
carcinoma cell line. Biochem Biophys Res Commun 198:
1281–1289.
2
7. Nutt RF, Caulfield MP, Levy JJ, Gibbons SW, Rosenblatt M,
McKee RL 1990 Removal of partial agonism from parathyroid
hormone (PTH)-related protein-(7–34)NH2 by substitution of 39. Rabbani SA, Gladu J, Liu B, Goltzman D 1995 Regulation in
PTH amino acids at positions 10 and 11. Endocrinology 127:
91–493.
8. Dunbar ME, Wysolmerski JJ 1999 Parathyroid hormone-
vivo of the growth of Leydig cell tumors by antisense ribonu-
cleic acid for parathyroid hormone-related peptide. Endocri-
nology 136:5416–5422.
4
2
related protein: A developmental regulatory molecule neces- 40. Akino K, Ohtsuru A, Yano H, Ozeki S, Namba H, Nakashima
sary for mammary gland development. J Mammary Gland Biol
Neoplasia 4:21–34.
9. Iezzoni JC, Bruns ME, Frierson HF, Scott MG, Pence RA,
Deftos LJ, Bruns DE 1998 Coexpression of parathyroid
hormone-related protein and its receptor in breast carcinoma:
A potential autocrine effector system. Mod Pathol 11:265–
M, Ito M, Matsumoto T, Yamashita S 1996 Antisense inhibi-
tion of parathyroid hormone-related peptide gene expression
reduces malignant pituitary tumor progression and metastases
in the rat. Cancer Res 56:77–86.
2
Address reprint requests to:
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2
70.
3
3
3
0. Downey SE, Hoyland J, Freemont AJ, Knox F, Walls J,
Bundred NJ 1997 Expression of the receptor for parathyroid
hormone-related protein in normal and malignant breast tissue.
J Pathol 183:212–217.
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(
995 Parathyroid hormone (PTH)/PTH-related protein
PTHrP) receptor expression and mitogenic responses in hu-
man breast cancer cell lines. Br J Cancer 72:90–95.
2. Guo J, Iida KA, Huang X, Abou-Samra A, Segre GV, Bring- Received in original form December 27, 1999; in revised form
hurst FR 1995 Parathyroid hormone (PTH)/PTH-related pep- April 24, 2000; accepted June 9, 2000.