Department of Medicine, McGill University, Montreal, Quebec H3A 1A1, Canada
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We examined parathyroid hormone-related peptide (PTHrP) production and regulation in both normal human melanocytes and in a human amelanotic melanoma cell line (A375). Northern blot and immunocytochemical analysis demonstrated that both cultured A375 cells and normal human melanocytes express PTHrP, but A375 cells expressed much higher levels of the peptide. PTHrP secretory rate increased at least 10-fold after treatment with 10% fetal bovine serum (100.2 ± 2.8 pmol/106 cells vs. basal <15 pmol/106 cells) in proliferating A375 cells but only twofold in confluent cells. Treatment of A375 cells with increasing concentrations of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] or its low-calcemic analog EB-1089 revealed that EB-1089 was 10-fold more potent than 1,25-(OH)2D3 on inhibition of both cell proliferation and PTHrP expression. Furthermore, inoculation of A375 cells into the mammary fat pad of female severe combined immunodeficiency mice resulted in the development of hypercalcemia and elevated concentrations of plasma immunoreactive PTHrP in the absence of detectable skeletal metastases. Our study, therefore, demonstrates a stepwise increase in PTHrP expression when cells progress from normal to malignant phenotype and suggests that EB-1089 should be further evaluated as a therapeutic agent in human melanoma.
malignant melanoma; vitamin D analog EB-1089
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
MALIGNANT MELANOMA CAN METASTASIZE to almost every major organ and tissue. In clinical series, osteolytic bone metastases have been recognized with a frequency ranging from 23 to 49% (4). Hypercalcemia is uncommon in this condition, and its pathogenesis remains uncertain (5, 31). As in other malignancies, it may be caused by local osteolytic mechanisms and/or by production by the tumor of biologically active substance(s), such as parathyroid hormone-related peptide (PTHrP) (14).
The human amelanotic melanoma A375 cell line was previously shown to induce osteolytic bone metastases and hypercalcemia when injected into the left cardiac ventricle of nude mice (17, 35). In addition, an antibody against PTHrP(1-34) blocked the formation of osteolytic bone lesions and the growth of metastatic deposits in a nude mouse model of bone metastases injected with a PTHrP-producing human breast cancer cell line (15), suggesting that PTHrP expression by breast cancer cells enhances their metastatic potential to bone. To gain further insight into the mechanism of tumor-induced osteolysis in malignant melanoma, we examined the expression and regulation of PTHrP production in normal and malignant melanocytes. In addition, we examined the in vivo production of PTHrP by the tumor and its calcemic effect after implantation of melanoma A375 cells into the mammary fat pad of athymic severe combined immunodeficiency (SCID) mice. Here, we demonstrated a stepwise increase in PTHrP expression when cells progress from normal to malignant melanocytes in vitro and its regulation by the vitamin D3 analog EB-1089. Furthermore, we show in vivo induction of hypercalcemia by the tumor in the absence of skeletal metastases.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Culture conditions. The human amelanotic malignant melanoma cell line A375 was established from a metastatic lesion in the lung (27). These cells metastasize to the lung at a high frequency when injected subcutaneously or intravenously into athymic nude mice or form skeletal metastases when injected into the left cardiac ventricle (17, 35).
A375 cells were obtained from American Type Culture Collection (Rockville, MD), maintained in Dulbecco's modified Eagle's medium (DMEM; GIBCO, Grand Island, NY) that contained 10% heat-inactivated fetal bovine serum (FBS; Wisent, Montreal, Quebec, Canada) and 1× antibiotic-antimycotic solution (GIBCO) at 37°C in an atmosphere of humidified air and 5% CO2, and passaged once weekly. Normal human epidermal melanocytes (NHEM) were obtained from Clonetics (San Diego, CA) and grown in a complete medium, melanocyte growth medium (MGM), consisting of melanocyte basal medium (MBM-2; Clonetics) supplemented with 1 ng/ml human basic fibroblast growth factor (bFGF; Clonetics), 5 µg/ml insulin, 0.5 µg/ml hydrocortisone, 10 ng/ml phorbol 12-myristate 13-acetate (Clonetics), 0.5% FBS, and 15 µg/ml bovine pituitary extract. For studies involving mRNA detection, cells were grown to 60% confluence in MGM. After a 24-h incubation in MBM-2 (basal conditions), the medium was changed and replaced with MGM for 4 h. Cells were then trypsinized and lysed with TRIzol solution (GIBCO) for subsequent analysis by Northern blot hybridization as described in Northern blot analysis.Cell proliferation assays.
A375 cells were seeded at a density of 1 × 104
cells/well in 24-well cluster plates and grown to 30% confluence.
After 24 h in serum-deprived DMEM, fresh medium that contained 1%
FBS with or without increasing concentrations (1010 to
10
7 M) of EB-1089 or 1,25-dihydroxyvitamin D3
[1,25-(OH)2D3] was added to cultured cells,
and incubations continued for 48-72 h. Ethanol concentration in
all cultures was 0.1%. Cells were trypsinized at timed intervals, an
aliquot was counted (Coulter Electronics, Beds, UK), and the remaining
cell suspension was spun and processed for Northern blot analysis.
Assay for immunoreactive PTHrP in conditioned medium.
An immunoradiometric assay (INCSTAR, Stillwater, MN) that employs two
polyclonal antibodies, one labeled with 125I that binds to
the COOH-terminal 57-80 region, and a second antibody that
recognizes the NH2-terminal 1-40 residue bound to a
solid phase, was used. The detection limit of the assay is 1.5 pmol/l. A375 cells were plated at a density of 1 × 104
cells/well in 24-well cluster plates and grown to 30% confluence. After 24 h in serum-deprived DMEM, cells were treated with
increasing concentrations of EB-1089 or
1,25-(OH)2D3 (1010 to
10
7 M), insulin (1-10 µg/ml), or bFGF (0.5-50
ng/ml). Conditioned medium was collected, centrifuged to remove debris,
and stored at
80°C until assayed. Cells were trypsinized and an
aliquot was counted. Results were corrected for cell number and
expressed as percentage of human PTHrP
[hPTHrP(1-84)]/106 cells in the
absence of 1,25-(OH)2D3 or its analog (% of control).
Northern blot analysis.
Total cellular RNA from cells or tumor tissues was isolated with TRIzol
solution according to the manufacturer's specifications (GIBCO),
dissolved in water, and stored at 80°C for future use. Twenty
micrograms of total RNA were electrophoresed in a 1.2% formaldehyde-agarose gel and transferred by blotting to a nylon membrane. Filters were air dried, baked at 80°C under vacuum for 2 h, and then prehybridized for at least 2 h at 65°C in a
solution of 5× SSC (1× SSC is 0.15 M sodium chloride, 0.015 M
trisodium citrate), 5× Denhardt's solution, 0.5% sodium dodecyl
sulfate (SDS), and denatured salmon sperm DNA. Hybridization was
carried out overnight at 65°C in the same buffer using a 537-bp
Sac I-Hind III restriction fragment encoding exon
III (coding region) of the hPTHrP gene labeled with
[32P]dCTP (ICN Biomedicals, Mississauga, Ontario, Canada)
using a random primer kit (Pharmacia Biotech, Baie d'Urfé,
Quebec, Canada). Filters were washed twice at room temperature in 2×
SSC and 0.1% SDS for 10 min and once for 15 min at 65°C in 1× SSC
and 0.1% SDS. Autoradiography of filters was carried out at
80°C using Kodak XAR film and two intensifying screens. Filters
were also probed with an 800-bp BamH I restriction fragment
of rat cyclophilin as a control for PTHrP mRNA changes.
RNA analysis by RT-PCR. Vitamin D receptor (VDR) was amplified using the following primers: VDR upstream primer, 5'-ATGGCGGCCAGCACTTCCCTGCCTGAC-3'; VDR downstream primer, 5'-CTCCTCCTTCCGCTTCAGGATCATCTC-5'. Briefly, 5 µg of total RNA were reverse transcribed using an RT mix consisting of 4 µl of 5× first-strand buffer (GIBCO BRL, Montreal, Quebec, Canada), 2 µl of deoxynucleotide triphosphate (10 mM), 1 µl of random hexamer primers, 0.25 µl of RNase inhibitor, 0.5 µl of RT, 0.4 µl of dithiothreitol (0.1 M), and 5.85 µl of water. Tubes were then placed in a thermocycler (Perkin-Elmer) and treated at the following temperatures: 10 min at 23°C, 45 min at 42°C, and 5 min at 95°C. A PCR mix consisting of 5 µl of 10× PCR buffer (GIBCO BRL), 2.5 µl (100 pmol) of each primer, 0.5 µl of Taq polymerase, and 34.5 µl of water was then added to 5 µl of RT product, and the DNA was amplified for 30 cycles. Analysis was performed on a 2% agarose gel stained with ethidium bromide.
Immunocytochemical analysis. Cellular content of PTHrP was determined by immunocytochemistry. Cells were seeded in four chamber glass slides (Nalge Nunc, Naperville, IL) at a density of 104 cells per chamber in either DMEM/10% FBS (A375 cells) or MGM (NHEM). Medium was changed 24 h before staining. Cells were then fixed in 1% Formalin in phosphate-buffered saline (PBS) for 15 min at 4°C, rinsed with PBS, and stained with a rabbit polyclonal antibody raised against hPTHrP(1-34) using a modification of the three-layer peroxidase antiperoxidase technique (28).
Animal protocols.
SCID female mice (Charles River, St-Constant, Quebec, Canada), weighing
13-16 g (3-4 wk old), were used in all studies. Approximately 1 × 106 A375 viable cells in 0.1 ml of FBS-free DMEM
were injected subcutaneously on the backs of animals with a 27-gauge
needle. To examine the effect of EB-1089 on tumor growth, osmotic
minipumps (model 2ML4; Alza, Palo Alto, CA) were implanted
subcutaneously, immediately adjacent to the tumor site, the same day as
the injection of A375 cells. Each minipump contained EB-1089 or vehicle
alone, dissolved in 50% propylene glycol, 10% ethanol, and 40%
saline, to deliver a continuous dose of the compound for up to 4 wk at
a delivery rate of 16 pM/24 h. This dose of EB-1089 given to
SCID mice was the maximal dose that did not induce hypercalcemia and
was tolerated without weight loss (data not shown). Each group
consisted of five mice. All animals were examined once a week for the
development of a palpable tumor at the site of injection.
Three-dimensional tumor measurements were done using calipers. Tumor
diameters, long axis (L), and mean midaxis width
(W), were measured to estimate the volume using the
following formula: 4/3(L/2×W/2)3.
A growth curve was generated by plotting the mean tumor volume over
time. Five weeks after tumor implantation, animals were killed, and
tumors were removed and weighed.
Plasma calcium and PTHrP measurements.
To examine the ability of A375 cells to develop hypercalcemia and
elevated serum immunoreactive PTHrP (iPTHrP), 1 × 106
viable semiconfluent A375 cells were injected through a 27-gauge needle
into the mammary fat pad (a single injection per mouse) of a SCID
female. Blood was collected by orbital bleeding for measurement of
total plasma calcium and albumin. Plasma calcium and albumin levels
were determined by microchemistry (Kodak Ektachrome, Mississauga,
Ontario, Canada). Corrected plasma calcium was calculated using the
following formula: plasma total calcium + (40 plasma albumin) × 0.02. Seven weeks after tumor cell
inoculation, mice were killed by cardiac puncture, and tumors were
excised and snap frozen for further RNA analysis. Plasma samples were
stored at
80°C until PTHrP analysis using an immunoradiometric
assay specific for PTHrP(1-86) (Diagnostic Systems
Laboratories, Webster, TX) as described previously (29).
Results were expressed as picogram equivalents of
PTHrP(1-86) per milliliter. The detection limit of
the assay was 2 pg equivalent of PTHrP(1-86)/ml plasma.
X-ray analysis of nude mice. Mice were anesthetized, placed in a prone position against the films (18 × 24 cm; AGFA, Mortsel, Belgium), and exposed to an X-ray at 25 kV for 5 s using a Mammo Diagnost UC (Philips, Hamburg, Germany). Films were developed using a Curix compact processor (AGFA). Radiographs were analyzed by three investigators, including one radiologist who had no knowledge of the experimental protocol.
Statistical analysis. All results are expressed as means ± SE of triplicate determinations, and statistical comparisons are based on one-way analysis of variance or Student's t-test. A probability value of P < 0.05 was considered significant.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Expression of PTHrP mRNA in human melanoma A375 cells, melanoma
tumor tissue, and NHEM.
PTHrP was seen as a single 1.6-kb transcript in both
FBS-treated melanoma A375 cells or growth factor-treated NHEM (Fig.
1). PTHrP expression in melanoma
A375 cells was at least 10 times higher than in normal melanocytes. The
data obtained in NHEM were done using melanocytes obtained from
different donors and were found similar. PTHrP was also detected in
melanoma tumor tissue produced by inoculation of A375 cells into the
mammary fat pad of athymic mice.
|
Immunocytochemical staining of normal and malignant human
melanocytes.
Immunocytochemical studies using a PTHrP antibody directed against
PTHrP(1-34) showed intense and widespread staining in
human melanoma A375 cells, compared with control cells treated with nonimmune serum (Fig. 2, A and
B). In contrast, the same PTHrP antibody stained only a
small number of normal human melanocytes (Fig. 2, C and
D), in keeping with the difference observed with PTHrP mRNA
expression.
|
PTHrP secretion in proliferating and confluent melanoma A375 cells
and in NHEM.
We then assessed iPTHrP release into the conditioned medium of A375
cells (Fig. 3). In proliferating
subconfluent A375 cells incubated with 10% FBS, there was a marked
(10-fold) increase of PTHrP secretory rate, compared with cells
incubated in the absence of FBS (basal secretory rate). In contrast,
when confluent cultures of A375 were treated in the same conditions,
FBS produced only a twofold increase in the secretory rate of PTHrP,
indicating that PTHrP secretion by A375 cells is much more pronounced
during the proliferative phase.
|
Effect of insulin and bFGF on PTHrP secretion in human melanoma
cells.
We subsequently analyzed the effect on PTHrP production of growth
factors previously shown to promote melanocyte cell proliferation. In
these experiments, cells were grown in DMEM in the absence (basal) or
presence (control) of a low-FBS (0.5%) concentration. The potential
additive effect of insulin (10 µg/ml) or bFGF (50 ng/ml) was analyzed
in the presence of 0.5% FBS. A small increase above basal level was
observed with 0.5% FBS (24 ± 0.5 vs. 20.1 ± 0.4, P < 0.05) but no further increase was seen with either insulin or bFGF (Table 1).
|
Effects of 1,25-(OH)2D3 and EB-1089 on the
proliferation of normal and malignant melanocytes.
We examined the effects of 1,25-(OH)2D3 and its
low-calcemic analog EB-1089 on proliferation of NHEM and melanoma A375
cells. These cells express VDR mRNA, as determined by RT-PCR of total RNA (data not shown). Addition of increasing concentrations of 1,25-(OH)2D3 or EB-1089 to the culture medium
of A375 cells caused a significant dose-dependent inhibition of cell
growth (Fig. 4). EB-1089 was, on average,
10-fold more potent than 1,25-(OH)2D3, as
determined by the concentration producing a 30% decrease of maximal
inhibition of cell growth (1010 M vs. 10
9
M) (Fig. 4A). Similar results were observed by assessing
inhibition of [3H]thymidine incorporation in EB-1089 or
1,25-(OH)2D3-treated cells (Fig.
4B). In addition, both 1,25-(OH)2D3
and EB-1089 produced a significant inhibition of normal melanocyte
growth, as assessed by formazan production (Fig. 4C).
|
Effects of 1,25-(OH)2D3 and EB-1089 on
PTHrP secretion.
Effects of increasing concentrations of
1,25-(OH)2D3 and EB-1089 (1010 to
10
7 M) on PTHrP secretion were next examined. Untreated
cells produced 75.5 ± 3.81 pmol/106 cells/24 h,
whereas both EB-1089 and 1,25-(OH)2D3 produced
a significant and dose-dependent inhibition of PTHrP secretion, similar
to the effects observed on cell growth (Fig.
5). EB-1089 was again, on average,
10-fold more potent than 1,25-(OH)2D3, as
assessed by the concentration of the hormone inducing a 30% inhibition
of PTHrP secretion. Results were corrected for cell number, indicating
that the observed effects were independent of the antiproliferative
action of EB-1089 and 1,25-(OH)2D3.
|
Effect of 1,25-(OH)2D3 and EB-1089 on PTHrP
mRNA expression in melanoma A375 cells.
EB-1089 and 1,25-(OH)2D3 effects on PTHrP mRNA
was examined by Northern blot analysis. Treatment of A375 cells with
increasing concentrations of 1,25-(OH)2D3 or
EB-1089 (109 to 10
7 M) for 3 h
produced a dose-dependent decrease in PTHrP mRNA levels (Fig.
6) with EB-1089 being more potent than
1,25-(OH)2D3 at 10
9 M and
10
8 M but not at 10
7 M.
|
In vivo analysis of calcium and PTHrP levels in SCID mice
inoculated into the mammary fat pad with A375 cells.
Palpable tumors developed within 10 days postinoculation, and animals
killed at 7 wk had metastases, primarily in the lymph nodes and lungs,
as determined by histological examination. However, no visible
osteolytic bone lesions were observed by X-ray analysis (data not
shown). Blood was collected in tumor-bearing animals at 7 wk and
demonstrated an increase both in plasma calcium and iPTHrP (Table
2). In contrast, PTHrP was undetectable
in nontumor-bearing mice.
|
Effect of EB-1089 on tumor growth and body weight.
After A375 cell implantation, palpable tumors occurred after the second
week and showed a rapid increase in volume until death of the SCID mice
at 5 wk (Fig. 7A). Three weeks
after tumor implantation, there was a significant tumor growth
inhibition in EB-1089-treated animals compared with the control group
(0.249 ± 0.098 vs. 0.411 ± 0.107 cm3;
n = 5, P < 0.05). At 5 wk, the mean
tumor volume in control mice was 2.5 ± 0.43 cm3,
compared with 1.43 ± 0.11 cm3 in EB-1089-treated
animals (P < 0.03). In addition, tumor weight, measured at death (5 wk), was significantly lower in EB-1089-treated mice (0.86 ± 0.12 g) than in control mice (1.4 ± 0.2 g; P < 0.04; Fig. 7B). In contrast
to the significant differences in tumor volume and weight, animal
weight was similar between the two groups for the duration of the
experiments (Fig. 7C).
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
A number of normal and cancer cells have previously been shown to
produce PTHrP (9, 12, 19, 20, 28, 30, 36, 38, 39). Studies
in normal cells (12, 18, 28) and knockout animals
(3, 26) indicate that PTHrP modulates cellular growth and
differentiation of numerous tissues, including skin (24, 25,
28), cartilage (3), and breast (39,
40). In the present study, we demonstrated that PTHrP is also
expressed at low levels in normal human melanocytes. One dominant
transcript was observed similar to what has been reported in other
cultured normal and cancer cell lines (10, 20, 28, 32).
Immunocytochemical analysis indicates that few normal human melanocytes
indeed express the peptide. However, PTHrP was undetectable in the
conditioned media of NHEM treated with growth factors, suggesting that
this peptide is unlikely to play an autocrine/paracrine function in this model. This low expression contrasts with the high expression observed in the human malignant melanoma A375 cells. In these cells,
PTHrP mRNA was at least 10-fold higher than in normal melanocytes. Furthermore, immunocytochemical analysis revealed that all cells expressed the peptide. The expressed PTHrP was also released in high
levels in the culture medium in vitro. These properties were also
observed in vivo after implantation of tumor cells into the mammary fat
pad. Animals grew tumors that rapidly metastasized to several
organs. mRNA extracted from tumors expressed high levels of
PTHrP, and the peptide was detected in the blood of animals that
rapidly developed hypercalcemia, indicating the endocrine effect of
PTHrP. A careful analysis of the metastatic properties of these cells
indicated that several major tissues were targeted. However, no obvious
osteolytic skeletal metastases were detected. It remains possible,
however, that small metastatic foci remain undetected on X-rays. In
previous studies (17, 35), the same cell line was injected
into the left ventricle of nude mice, inducing the rapid development of
skeletal metastases with concomitant hypercalcemia. The authors
postulated that transforming growth factor- overexpression by A375
tumor cells could be responsible for tumor-induced osteolysis and
subsequent hypercalcemia. However, PTHrP was not measured in the blood
of the animals or in the tumor cells within bone.
Although uncommon, hypercalcemia has been reported in patients with human melanoma with or without bone metastases, but its pathogenesis remains uncertain. In some studies, hypercalcemic patients with melanoma were reported to have suppressed levels of parathyroid hormone but elevated levels of nephrogenous cAMP excretion (5, 13, 31), which suggests a possible link with PTHrP production by melanoma cells. Recently, several studies have reported the occurrence of hypercalcemia in patients with malignant melanoma and identified PTHrP as one of the causative factors of hypercalcemia (23, 34, 41). Osteolytic bone metastases are also a manifestation of malignant melanoma. However, as postulated for breast cancer (14, 15, 42), PTHrP may contribute to the pathogenesis of osteolytic bone destruction in this condition. Further studies will be required to determine whether PTHrP contributes to the development of bone metastases in this melanoma model.
The biologically active metabolite of vitamin D3, 1,25-(OH)2D3, has properties that extend beyond those of regulating bone mineralization and calcium homeostasis. Previous in vitro and in vivo studies have clearly demonstrated that 1,25-(OH)2D3 is a potent antiproliferative agent and suggest a possible clinical use of this hormone in the treatment of hyperproliferative disorders. However, its clinical usefulness is restricted by its strong calcemic activity. Consequently, the search for new vitamin D analogs with more potent growth inhibitory properties but reduced calcemic activity has intensified in the past several years. Among these analogs, EB-1089, characterized by an ethyl group and double bonds in the side chain and a half-life similar to 1,25-(OH)2D3 in vivo (33), has been studied extensively. This compound has more potent antiproliferative activity than 1,25-(OH)2D3 while being 50% as calcemic as its parent compound (2, 7, 8, 16, 22, 37, 44). In the present study, we show that the vitamin D analog EB-1089, as well as 1,25-(OH)2D3, inhibits melanoma A375 cell growth and PTHrP expression and secretion. Furthermore, EB-1089 is more potent than the native hormone by at least one order of magnitude. These results are consistent with the reported observations that PTHrP is negatively regulated by 1,25-(OH)2D3 and its synthetic analogs in several normal and cancer cells, including cultured normal human mammary cells (36), cultured human keratinocytes (28, 44), human lung squamous cancer cells (11), MT-2 cells (a cell line derived from human T cell leukemia virus I-infected T cells) (21), and oral cancer cells (1). In addition to these effects in vitro, our data also indicate that EB-1089 significantly reduces the growth of melanoma A375 cells in vivo. This effect occurs without significant calcium elevation and body weight loss. Several in vivo studies have previously demonstrated the efficacy of EB-1089 in reducing the growth of a variety of malignancies without affecting serum calcium levels (15, 18, 28-30), indicating that EB-1089 has selective properties on target tissues and, particularly, cancer cells at a dose that has no apparent effect on calcium homeostasis. In view of the fact that the VDR is expressed in a wide variety of cancer cells, including human malignant melanoma cells (6), EB-1089 may prove to be an interesting adjuvant in the chemotherapeutic arsenal against human melanoma.
Because normal human melanocytes require specific growth factors, such as bFGF, to proliferate in culture, and because bFGF is one of the most abundant growth factors in the bone matrix (43), we assessed the influence of bFGF on PTHrP release from melanoma A375 cells. The bFGF effect was analyzed in various conditions both in the absence and in the presence of FBS over several days and did not significantly modulate either cellular proliferation or the secretion of PTHrP into the conditioned medium. The absence of a bFGF effect is likely due to an absent or inactive bFGF receptor in this model. Finally, we examined the effect of insulin on both cellular proliferation and PTHrP secretion. Although insulin was a potent mitogen in this system, we could not demonstrate any effect of insulin on PTHrP secretion. The absence of an insulin effect in this study is similar to our previous report on normal human keratinocytes (28) but contrasts with the well-described influence of insulin on modulating PTHrP secretion reported in normal human mammary epithelial cells (36), suggesting that PTHrP stimulation by mitogens is regulated in a tissue-specific fashion.
In conclusion, our results indicate that PTHrP is expressed in both melanocytes and the human melanoma cell line A375 and that its high level of expression in the latter model is closely associated with the development of hypercalcemia in the absence of osteolytic bone lesions. The potent action of EB-1089 on inhibition of both cell proliferation and PTHrP expression may provide a new strategy for the treatment of this common malignancy.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. Lise Binderup (Leo Pharmaceuticals) for kindly providing EB-1089.
![]() |
FOOTNOTES |
---|
This work was supported by a grant from The Dairy Farmers of Canada (to R. Kremer) and the Medical Research Council of Canada Grants MT 10839 (to R. Kremer) and MT 5775 (to D. Goltzman).
Address for reprint requests and other correspondence: R. Kremer, Calcium Research Lab, Rm. H4.67, Royal Victoria Hospital, 687 Pine Ave. W., Montreal, Quebec H3A 1A1, Canada.
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 29 November 1999; accepted in final form 3 May 2000.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Abe, M,
Akeno N,
Ohida S,
and
Horiuchi N.
Inhibitory effects of 1,25-dihydroxyvitamin D3 and 9-cis-retinoic acid on parathyroid hormone-related protein expression by oral cancer cells (HSC-3).
J Endocrinol
156:
349-357,
1998
2.
Akhter, J,
Goerdel M,
and
Morris DL.
Vitamin D3 analog (EB1089) inhibits in vitro cellular proliferation of human colon cancer cells.
Br J Surg
83:
229-230,
1996[ISI][Medline].
3.
Amizuka, N,
Warshawsky H,
Henderson JE,
Goltzman D,
and
Karaplis AC.
Parathyroid hormone-related peptide-depleted mice show abnormal epiphyseal cartilage development and altered endochondral bone formation.
J Cell Biol
126:
1611-1623,
1994[Abstract].
4.
Balch, CM,
and
Milton GW.
Diagnosis of metastatic melanoma at distant sites.
In: Cutaneous Melanoma: Clinical Management and Treatment Results Worldwide, edited by Balch CM,
and Milton GW.. Philadelphia, PA: Lippincott, 1985, p. 221.
5.
Burt, ME,
and
Brennan MF.
Hypercalcemia and malignant melanoma.
Am J Surg
137:
790-794,
1979[ISI][Medline].
6.
Colston, KW,
Colston MJ,
and
Feldman D.
1,25-Dihydroxyvitamin D3 and malignant melanoma: the presence of receptors and inhibition of cell growth in culture.
Endocrinology
108:
1083-1086,
1981[Abstract].
7.
Colston, KW,
James SY,
Ofori-Kuragu EA,
Binderup L,
and
Grant AG.
Vitamin D receptors and anti-proliferative effects of vitamin D derivatives in human pancreatic carcinoma cells in vivo and in vitro.
Br J Cancer
76:
1017-1020,
1997[ISI][Medline].
8.
Colston, KW,
Mackay AG,
James SY,
Binderup L,
Chander S,
and
Coombes RC.
EB1089: a new vitamin D analogue that inhibits the growth of breast cancer cells in vivo and in vitro.
Biochem Pharmacol
44:
2273-2280,
1992[ISI][Medline].
9.
Deftos, LJ,
Gazdar AF,
Ikeda K,
and
Broadus AE.
The parathyroid hormone-related protein associated with malignancy is secreted by neuroendocrine tumors.
Mol Endocrinol
3:
503-598,
1989[Abstract].
10.
Drucker, DJ,
Asa SL,
Henderson J,
and
Goltzman D.
The parathyroid hormone-like peptide gene is expressed in the normal and neoplastic human endocrine pancreas.
Mol Endocrinol
3:
1589-1595,
1989[Abstract].
11.
Falzon, M.
The noncalcemic vitamin D analogues EB1089 and 22-oxacalcitriol interact with the vitamin D receptor and suppress parathyroid hormone-related peptide gene expression.
Mol Cell Endocrinol
127:
99-108,
1997[ISI][Medline].
12.
Ferrari, SL,
Rizzoli R,
and
Bonjour JP.
Parathyroid hormone-related protein production by primary cultures of mammary epithelial cells.
J Cell Physiol
150:
304-311,
1992[ISI][Medline].
13.
Green, G,
and
Veitch P.
Amelanotic melanoma and hypercalcemia.
Aust NZ J Med
26:
716-717,
1996[ISI][Medline].
14.
Grill, V,
Rankin W,
and
Martin TJ.
Parathyroid hormone-related protein (PTHrP) and hypercalcaemia.
Eur J Cancer
34:
222-229,
1998[ISI][Medline].
15.
Guise, TA,
Yin JJ,
Taylor SD,
Kumagai Y,
Dallas M,
Boyce BF,
Yoneda T,
and
Mundy GR.
Evidence for a causal role of parathyroid hormone-related protein in the pathogenesis of human breast-mediated osteolysis.
J Clin Invest
98:
1544-1549,
1996
16.
Hansen, CM,
and
Maenpaa PH.
EB1089, a novel vitamin D analog with strong antiproliferative and differentiation-inducing effects on target cells.
Biochem Pharmacol
54:
1173-1179,
1997[ISI][Medline].
17.
Hiraga, T,
Nakajima T,
and
Ozawa H.
Bone resorption induced by a metastatic human melanoma cell line.
Bone
16:
349-356,
1995[ISI][Medline].
18.
Holick, MF,
Ray S,
Chen TC,
Tian X,
and
Persons KS.
A parathyroid hormone antagonist stimulates epidermal proliferation and hair growth in mice.
Proc Natl Acad Sci USA
91:
8014-8016,
1994[Abstract].
19.
Hongo, TJ,
Kupfer J,
Enomoto H,
Sharifi B,
Giannella-Neto D,
Forrester JS,
Singer FR,
Goltzman D,
Hendy GN,
Pirola C,
Fagin JA,
and
Clemens TL.
Abundant expression of parathyroid hormone related protein in primary rat aortic smooth muscle cells accompanies serum induced proliferation.
J Clin Invest
88:
1841-1847,
1991[ISI][Medline].
20.
Ikeda, K,
Weir EC,
Mangin M,
Dannies PS,
Kinder B,
Deftos LJ,
Brown EM,
and
Broadus AE.
Expression of messenger ribonucleic acids encoding a parathyroid hormone-like peptide in normal human and animal tissues with abnormal expression in human parathyroid adenomas.
Mol Endocrinol
2:
1230-1236,
1998[Abstract].
21.
Inoue, D,
Matsumoto T,
Ogata E,
and
Ikeda K.
22-Oxacalcitriol, a noncalcemic analogue of calcitriol, suppresses both cell proliferation and parathyroid hormone-related peptide gene expression in human T cell lymphotrophic virus, type I-infected T cells.
J Biol Chem
268:
16730-16736,
1993
22.
James, SY,
Mackay AG,
Binderup L,
and
Colston KW.
Effects of a new synthetic vitamin D analogue, EB1089, on the estrogen-responsive growth of human breast cancer cells.
J Endocrinol
141:
555-563,
1994[Abstract].
23.
Kageshita, T,
Matsui T,
Hirai S,
Fukuda Y,
and
Ono T.
Hypercalcaemia in melanoma patients associated with increased levels of parathyroid hormone-related protein.
Melanoma Res
9:
69-73,
1999[ISI][Medline].
24.
Kaiser, SM,
Laneuville P,
Bernier SM,
Rhim JS,
Kremer R,
and
Goltzman D.
Enhanced growth of a human keratinocyte cell line induced by antisense RNA for parathyroid hormone-related peptide.
J Biol Chem
267:
13623-13628,
1992
25.
Kaiser, SM,
Sebag M,
Rhim JS,
Kremer R,
and
Goltzman D.
Antisense-mediated inhibition of parathyroid hormone-related peptide production in a keratinocyte cell line impedes differentiation.
Mol Endocrinol
8:
139-147,
1994[Abstract].
26.
Karaplis, AC,
Luz A,
Glowacki J,
Bronson RT,
Tybulewicz VL,
Kronenberg HM,
and
Mulligan RC.
Lethal skeletal dysplasia from targeted distruption of the parathyroid hormone-related peptide gene.
Genes Dev
8:
277-289,
1994[Abstract].
27.
Kozlowski, JM,
Hart IR,
Fidler IJ,
and
Hanna N.
A human melanoma line heterogeneous with respect to metastatic capacity in athymic nude mice.
J Natl Cancer Inst
72:
913-917,
1984[ISI][Medline].
28.
Kremer, R,
Karaplis AC,
Henderson J,
Gulliver W,
Banville D,
Hendy GN,
and
Goltzman D.
Regulation of parathyroid hormone-like peptide in cultured normal human keratinocytes. Effect of growth factors and 1,25-dihydroxyvitamin D3 on gene expression and secretion.
J Clin Invest
87:
884-893,
1991[ISI][Medline].
29.
Kremer, R,
Shustik C,
Tabak T,
Papavasiliou V,
and
Goltzman D.
Parathyroid hormone related peptide (PTHRP) in hematologic malignancies.
Am J Med
100:
406-411,
1996[ISI][Medline].
30.
Kremer, R,
Woodworth CD,
and
Goltzman D.
Expression and action of parathyroid hormone-related peptide in human cervical epithelial cells.
Am J Physiol Cell Physiol
271:
C164-C171,
1996
31.
Levy, I,
and
Feun L.
Hypercalcemia and malignant melanoma.
Am J Clin Oncol
13:
524-526,
1990[ISI][Medline].
32.
Mangin, M,
Ikeda K,
Dreyer BE,
Milstone L,
and
Broadus AE.
Two distinct tumor-derived parathyroid hormone-like peptides result from alternative ribonucleic acid splicing.
Mol Endocrinol
2:
1049-1055,
1988[Abstract].
33.
Mathiasen, IS,
Colston KW,
and
Binderup L.
EB1089, a novel vitamin D analogue, has strong antiproliferative and differentiation inducing effects on cancer cells.
J Steroid Biochem Mol Biol
46:
365-371,
1993[ISI][Medline].
34.
Matsui, T,
Kageshita T,
Ishihara T,
Tomiguchi S,
Takahashi M,
and
Ono T.
Hypercalcemia in a patient with malignant melanoma arising in congenital giant pigmented nevus. A case of increased serum level of parathyroid hormone-related protein.
Dermatology
197:
65-68,
1998[ISI][Medline].
35.
Nakai, M,
Mundy GR,
Williams PJ,
Boyce B,
and
Yoneda T.
A synthetic antagonist to laminin inhibits the formation of osteolytic metastases by human melanoma cells in nude mice.
Cancer Res
52:
5395-5399,
1992[Abstract].
36.
Sebag, M,
Henderson J,
Goltzman D,
and
Kremer R.
Regulation of parathyroid hormone-related peptide production in normal human mammary epithelial cells in vitro.
Am J Physiol Cell Physiol
267:
C723-C730,
1994
37.
Skowronski, RJ,
Peehl DM,
and
Feldman D.
Actions of vitamin D3, analogs on human prostate cancer cell lines: comparison with 1,25-dihydroxyvitamin D3.
Endocrinology
136:
20-26,
1995[Abstract].
38.
Strewler, GJ,
Stern PH,
Jacobs JW,
Eveloff J,
Klein RF,
Leung SC,
Rosenblatt M,
and
Nissenson RA.
Parathyroid hormone-like protein from human renal carcinoma cells.
J Clin Invest
80:
1803-1897,
1987[ISI][Medline].
39.
Thiede, M,
and
Rodan GA.
Expression of calcium-mobilizing parathyroid hormone-like peptide in lactating mammary tissue.
Science
242:
278-280,
1988[ISI][Medline].
40.
Wysoolmerski, JJ,
McCaugheern CJ,
Daaiffotis AG,
Broadus AE,
and
Philbrick WM.
Overexpression of parathyroid hormone-related protein or parathyroid hormone in transgenic mice impairs branching morphogenesis during mammary gland development.
Development
121:
3539-3547,
1995
41.
Yeung, SC,
Eton O,
Burton DW,
Deftos LJ,
Vassilopoulou-Sellin R,
and
Gagel RF.
Hypercalcemia due to parathyroid hormone-related protein secretion by melanoma.
Horm Res
49:
288-291,
1998[ISI][Medline].
42.
Yoneda, T.
Cellular and molecular mechanisms of breast and prostate cancer metastasis to bone.
Eur J Cancer
34:
240-245,
1998[ISI][Medline].
43.
Yoneda, T,
Sasaki A,
and
Mundy GR.
Osteolytic bone metastasis in breast cancer.
Breast Cancer Res Treat
32:
73-84,
1994[ISI][Medline].
44.
Yu, J,
Papavasiliou V,
Rhim J,
Goltzman D,
and
Kremer R.
Vitamin D analogs: new therapeutic agents for the treatment of squamous cancer and its associated hypercalcemia.
Anticancer Drugs
6:
101-108,
1995[ISI][Medline].
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Visit Other APS Journals Online |