Endocrinology Vol. 138, No. 5 1879-1885
Copyright © 1997 by The Endocrine Society
The Role of Interleukin-6 in the Induction of Hypercalcemia in Renal Cell Carcinoma Transplanted into Nude Mice
Max G. Weissglas,
Denis H. J. Schamhart,
Clemens W. G. M. Löwik,
Socrates E. Papapoulos,
Harry M. Theuns and
Karl-Heinz Kurth
Departments of Urology (M.G.W.) and Endocrinology (C.W.G.M.L.,
S.E.P.), University Hospital Leiden, and the Division of Vascular and
Connective Tissue Research, TNO-Prevention and Health (H.M.T.), Leiden;
and the Department of Urology (D.H.J.S., K.-H.K.), University of
Amsterdam, Amsterdam, The Netherlands
Address all correspondence and requests for reprints to: Dr. M. G. Weissglas, University Hospital Leiden, Department of Urology, J3-P, P.O. Box 9600, 2300 RC Leiden, The Netherlands.
 |
Abstract
|
|---|
Hypercalcemia is a well known complication of renal cell carcinoma
(RCC). As RCCs can produce IL-6, and IL-6 may stimulate bone resorption
and cause mild hypercalcemia, we examined whether IL-6 is involved in
renal cancer-associated hypercalcemia in vivo. Three
human renal cell carcinoma tumor lines (RC-8, RC-9, and NC-65) growing
in nude mice were studied. Tumors were implanted sc, and parameters of
bone metabolism and serum human IL-6 levels were determined in relation
to tumor volume (TV). All three tumor lines secreted human IL-6,
although in different quantities. The maximum level of IL-6 in RC-8 was
434 pg/ml (TV, 200 mm3), that in RC-9 was 81 pg/ml (TV,
1800 mm3), and that in NC-65 was 2368 pg/ml (TV, 1800
mm3). Hypercalcemia developed in RC-8 and RC-9
tumor-bearing animals, but not in NC-65-bearing animals. The
hypercalcemia in both RC-8 and RC-9 tumor lines was associated with
elevated levels of PTH-related peptide (PTHrP) and loss of trabecular
bone volume. Serum calcium and phosphate concentrations showed an
almost linear relationship with plasma PTHrP independently of the tumor
line and serum IL-6 levels. No hypercalcemia occurred in the NC-65
animals, which had the highest levels of IL-6, but no detectable plasma
PTHrP and PTHrP messenger RNA expression in the tumor. Administration
of neutralizing antibodies to IL-6 to RC-8 animals normalized serum
calcium concentrations and PTHrP values and induced a significant
inhibition of tumor growth. No such effect on tumor growth of anti-IL-6
was seen in the other two tumor lines. The normalization of serum
calcium in RC-8 mice is most likely attributed to the growth-inhibiting
effect of anti-IL-6 on RC-8 tumor. We conclude that IL-6 secreted by
RCC does not contribute directly to hypercalcemia, but may enhance
hypercalcemia by stimulating the tumor growth of a subpopulation of
PTHrP-secreting carcinomas.
|
Introduction
|
|---|
CANCER-ASSOCIATED hypercalcemia is
primarily the result of increased bone resorption by humoral factors
secreted by the tumors (1). Hypercalcemia is a well known
paraneoplastic syndrome in renal cell carcinoma (RCC). PTH-related
peptide (PTHrP) is recognized as the major humoral factor responsible
for the induction of hypercalcemia, but other factors acting on bone
may also contribute to the increase in serum calcium concentrations
(2). One such factor is interleukin-6 (IL-6) (3, 4, 5). Renal cell
carcinomas have been reported to produce IL-6 in vitro (6)
and to express IL-6 messenger RNA (mRNA) and IL-6 receptor mRNA
in vivo (7, 8). It is not clear, however, whether IL-6
secreted by the tumor has any biological effect. It is possible that
IL-6 may act synergistically with PTHrP to cause hypercalcemia in RCC,
as has been described in a patient with pheochromocytoma (9) and in
nude mice inoculated with Chinese hamster ovarian cells transfected
with complementary DNAs (cDNAs) for PTHrP and IL-6 (10). In an earlier
study we reported cosecretion of IL-6 and PTHrP by a renal cell
carcinoma that caused hypercalcemia after implantation into nude mice
(11). After administration of neutralizing antibodies to IL-6, serum
calcium concentrations fell to almost normal, suggesting a contributing
role of IL-6 to the development of hypercalcemia in this tumor.
To examine further the role of IL-6 in hypercalcemia and its
interaction with PTHrP, we studied tumor parameters, biochemical and
histological indiexes of calcium and bone metabolism, and secretion of
human IL-6 (hIL-6) and PTHrP in nude mice inoculated with three
different renal cell carcinoma tumor lines, two of which induced
hypercalcemia.
|
Materials and Methods
|
|---|
Tumor lines
The three tumor lines, RC-8, RC-9, and NC-65, have all
been derived from patients with renal cell carcinomas in the advanced
stage and have been maintained in nude mice (12, 13, 14, 15). During subsequent
passages, no alterations occurred in the growth kinetics, histology, or
immunohistochemistry of the tumors. The donor patient of tumor line
RC-8 had hypercalcemia before operation; no information was available
about the serum calcium concentrations of the other two patients.
Animals
Four- to 6-week-old Swiss nu/nu mice were housed in a
laminar flow cage rack and were maintained at controlled temperature
(25 C) and humidity (60%). Acidified water was given ad
libitum, and (irradiated) standard rodent chow RMH-GS (Hope Farms,
Bodegraven, The Netherlands) containing 0.84% calcium was
provided.
Study protocol
Selected tumor pieces (2 x 2 x 2 mm3)
were sc inserted unilaterally into the shoulder region of
ether-anesthetized mice. Animals with a tumor volume (TV) of 100
mm3 were selected and divided into groups of five mice. TV
was assessed by measuring the two major diameters by the formula:
TV = 
/6(d1 x d2)1.5. At
different TVs, predefined on the basis of pilot experiments, animal
weight was recorded, and blood was taken for the determination of
biochemical parameters of calcium metabolism, PTHrP and IL-6. The blood
of all animals per TV of each tumor line was pooled because that
obtained from individual mice was not sufficient for the simultaneous
measurement of all metabolic parameters; this was a problem,
particularly in the hypercalcemic cachectic mice. Each group of animals
was subsequently killed, femurs were excised for bone histomorphometry,
and the tumor was removed for determination of PTHrP mRNA and IL-6
mRNA.
Neutralizing antibodies to IL-6 were provided by Dr. Lucien Aarden
(CLB, Amsterdam, The Netherlands). The neutralizing antibody to IL-6
(CLB IL/8, isotype IgGI kappa, Kd = 10-11)
neutralizes recombinant IL-6 and natural IL-6 equally well (16) and was
used in a concentration of 1.5 mg dissolved in 1 ml PBS (1 mg/mouse).
This dose can effectively neutralize all circulating hIL-6 measured in
the hypercalcemic mice (Dr. L. Aarden, personal communication).
Anti-IL-6 was administered by a single ip injection at a TV of
approximately 100 mm3 (n = 5–9). After 11 days, blood
was taken, and the animals were killed. This time frame was chosen as
hypercalcemia was expected to develop during that period of tumor
growth in RC-8 and RC-9 tumor-bearing animals and to prevent possible
dissociation of IL-6 from accumulated immune complexes.
Biochemical determinations
Serum calcium and phosphate concentrations were determined by
automated autoanalyzer techniques. Serum IL-6 was measured by an
enzyme-linked immunosorbent assay specific for hIL-6 (Medgenix,
Amersfoort, The Netherlands). Mouse IL-6 did not cross-react with the
hIL-6 enzyme-linked immunosorbent assay.
Plasma PTHrP was measured by immunoradiometric assay (Nichols
Institute, San Juan Capistrano, CA), using an 125I-labeled
anti-PTHrP 1–40 (sheep) and anti-PTHrP 60–72 (goat) with
PTHrP-(1–86) as standard. The detection limit of the assay was 0.7
pmol/liter, with an interassay variance of less than 6% at several
levels.
Northern blot analysis
After excision, tumor material was directly frozen and kept at
-70 C until RNA isolation. Total cellular RNA from tumors was isolated
by grinding (1 min; Polytron, Kinematica, Kriens/Luzern, Switzerland)
in ice-cold lithium chloride (3.3 M)-ureum (6.6
M). After overnight incubation at -20 C, the homogenate
was centrifuged at 10,000 x g for 30 min at 4 C, and
the pellet was resuspended in 10 mM Tris-0.5% SDS, pH 7.7.
Total RNA was extracted with three cycles of phenol-chloroform-isoamyl
alcohol (25:24:1) and subsequently precipitated at -20 C in 70%
ethanol-0.1 M sodium acetate, pH 5.2. The pellet was
resuspended in 10 mM Tris-1 mM EDTA, pH 7.6.
Total RNA was quantified by spectrophotometry at 260 nm. RNA samples
were analyzed using electrophoresis on a 1% denatured agarose gel
containing 7.5% formaldehyde and transferred to a nylon membrane
(Hybond N, Amersham, Aylesbury, UK). The membranes were hybridized with
32P-labeled probes specific for PTHrP (kindly provided by
M. Karperien, Hubrecht Laboratories, Utrecht, The Netherlands) and
hIL-6 cDNA [an 819-bp hIL-6 probe prepared using the reverse
transcriptase-PCR (RT-PCR) technique according to the method of
Kaashoek et al. (17), kindly provided by R. A. de Paus,
Department of Hematology, University Hospital Leiden, The Netherlands]
at 60 C in 7% SDS, 0.5 M NaHPO4 (pH 7.2), and
10 mM EDTA. 28S ribosomal RNA (rRNA) was used as an
internal control (kindly provided by Dr. C. Backendorf, Gorlaeus
Laboratory, Leiden, The Netherlands). As a positive control for PTHrP
and IL-6, total RNAs from the squamous cell carcinoma cell line SCC-4
and from IL-6-stimulated human fibroblasts (18) were used. The blots
were washed with 2 x SSC (0.30 M NaCl and 0.031
M sodium citrate) and 1% SDS for 60 min at 60 C.
RT-PCR
Semiquantitative RT-PCR was performed in a single reaction tube.
In this experimental set-up, the RT reaction and the subsequent PCR
were performed in a single 0.5-ml reaction tube. RNA was linearized by
heating for 5 min at 70 C, followed by quick chilling on ice. cDNA was
synthesized in a 10- or 20-µl reaction volume containing mRNA to be
reverse transcribed, PCR buffer [10 mM Tris-HCl (pH 9.6),
50 mM NaCl, and 0.2 mg BSA/ml], 5 mM
MgCl2, 1.5 mM deoxy (d)-GTP/dCTP/dATP/dTTP, 1 U
RNasin (Promega, Leiden, The Netherlands)/µl, 200 ng random
hexanucleotide primers (Promega, Leiden, The Netherlands)/µg RNA, and
2.5 U Moloney murine leukemia virus RT (Life Technologies, Paisley,
UK)/µl. Preparation of the RT mixture and addition of RNA to the RT
mixture were performed on ice to minimize RNase activity. To obtain
homogeneity, tubes were carefully vortexed and spun. Mixture was
overlayed with two or three drops of light white mineral oil (Sigma) to
reduce evaporation. As random hexamers were used, all tubes were
incubated for 10 min at room temperature to extend the hexameric
primers by RT, allowing the hexameric primers to remain annealed to the
RNA template upon raising the reaction temperature to 42 C. The RT
reaction was carried out by subsequently incubating all samples for 15
min at 42 C, 5 min at 99 C, and 5 min at 25 C with a Hybaid Omnigene
thermal cycler (Biozym, Landgraaf, The Netherlands). In all
experiments, the presence of possible contaminants was checked by a
control reaction in which RT-PCR was carried out on a sample in which
autoclaved denatured water instead of RNA was added to the RT reaction.
The subsequent amplification process was performed in the same reaction
tube in a final reaction volume of 50 or 100 µl containing PCR
buffer, 2 mM MgCl2, 0.2 µM of
each sense and anti-sense primers, 2.5 mU Super Thermus thermophilus
(S-Tth) DNA polymerase (HT Biotechnology, Cambridge, UK)/ml, and the
10- or 20-µl RT mixture, respectively. To obtain homogeneity, all
tubes were carefully vortexed and spun. After one cycle of 2 min at 95
C, the samples were amplified by repeated cycles of 30 sec at 95 C, 30
sec at 60 C, and 1 min at 72 C, followed by one cycle of 7 min at 72 C.
The samples were then held at 25 C. The annealing and extension
temperature of 72 C was not adjusted to the use of different primer
sets, unless otherwise specified. Ten-microliter aliquots of each
amplified sample were subjected to electrophoresis on 1% or 2%
agarose gels containing 0.5 µg ethidium bromide/ml in electrophoresis
buffer (44.5 mM Tris, 44.5 mM boric acid, and 1
mM EDTA) and photographed or stored at 4 C until subsequent
analysis.
Bone histomorphometry
From each mouse one femur was cleaned of soft tissue. The bones
were fixed in 10% neutral buffered formalin, slightly trimmed,
dehydrated in an ascending series of ethanol, infiltrated in
methylmethacrylate (MMA), and embedded in MMA. After polymerization,
the MMA blocks were trimmed and cut using a heavy duty microtome (HM
350, Microm, Heidelberg, Germany). Sections (4 µm) were stained
according to the method of von Kossa, which colors mineralized bone
black. The trabecular bone volume (BV/TV) (19) was measured using the
Optimas histomorphometric package (Bioscan Inc., Edmonds, WA) attached
to a Nikon microphot FXA microscope (Melville, NY) and an MX5 CCD
camera (Adimex Image Systems BV, Eindhoren, The Netherlands). All
measurements were made at a distance of approximately 0.5–1.0 mm from
the epiphyseal plate.
Statistical analysis
Tumor growth rates, analyzed per tumor, were calculated from the
slopes obtained by linear regression analysis. In tests for differences
between the various groups, series of one-way ANOVAs with equal and
unequal sizes were used. When significant (P < 0.05)
differences were found by ANOVA, Duncan’s multiple range test (20) or,
in the case of an unequal number of replications, Duncan’s multiple
range test adjusted by Kramer (21) was performed (P =
0.05). In cases of two conditions, differences were analyzed by
two-sided Student’s t test.
|
Results
|
|---|
Growth characteristics
Tumors were developed in nude mice after sc implantation of all
three tumor lines. The RC-8 tumors displayed the lowest growth rate and
reached a much lower TV due to death of the animals at TVs between
400–500 mm3 associated with extreme cachexia. In animals
implanted with RC-9 tumors, there was also a significant loss of
weight, which, however, occurred at a much later stage and at higher
TVs (1000 mm3). NC-65 tumors could be maintained in the
animals without any weight loss until a very high tumor load was
achieved (2000 mm3; Table 1
).
View this table:
[in this window]
[in a new window]
|
Table 1. Characteristics and biochemical parameters in nude
mice implanted with three different renal cell carcinoma tumor lines
|
|
Hypercalcemia
After transplantation, two of the three tumor lines, RC-8 and
RC-9, induced severe hypercalcemia (to a maximum of 6.24 and 7.22
mmol/liter, respectively) and hypophosphatemia (to a minimum of 1.55
and 1.37 mmol/liter, respectively; Table 1
). The degree of
hypercalcemia and hypophosphatemia was similar in animals bearing these
two tumors; there was, however, a pronounced difference in the TV
associated with these levels (469 ± 31.3 mm3 in RC-8
vs. 1827 and 1838 mm3 in RC-9). Generally, the
RC-8 tumor induced hypercalcemia of a degree comparable to that of RC-9
at approximately half the TV. Hypercalcemia and hypophosphatemia did
not occur in NC-65 tumor-bearing animals up to TVs of 1855 ± 116
mm3.
Serum levels of IL-6
After transplantation into nude mice, all three tumor lines
produced hIL-6 (Table 1). Serum IL-6 levels increased initially with
tumor growth in RC-8 and NC-65 tumor-bearing animals, reaching a
plateau, whereas there was a small increase in RC-9-bearing animals.
There were wide variations among the different tumor lines. The NC-65
tumor line produced the highest amounts of IL-6 (maximum, 2368 pg/ml),
and RC-9 produced the lowest amounts (maximum, 80 pg/ml), whereas the
IL-6 levels in RC-8 were intermediate (maximum, 434 pg/ml). There was
no relation between serum IL-6 levels and serum calcium
concentrations.
Plasma PTHrP levels
Both RC-8 and RC-9 induced comparable rises in plasma PTHrP to
maximums of 20.1 and 22.1 pmol/liter, respectively. In contrast, plasma
PTHrP remained undetectable in the animals implanted with the NC-65
tumor. The levels of PTHrP increased with TV in the RC-8- and
RC-9-implanted animals, but the RC-8 tumors secreted higher amounts per
unit volume. There existed a significant correlation between serum
calcium and phosphate concentrations and plasma PTHrP levels regardless
of tumor line (r = 0.971; P < 0.001 and r =
0.962; P < 0.001, respectively; Fig. 1).
View larger version (17K):
[in this window]
[in a new window]
|
Figure 1. Relation between plasma PTHrP levels and serum
calcium concentrations (upper panel) and serum phosphate
concentrations (lower panel) in nude mice bearing RC-8
( ) and RC-9 (•) tumors. Each point represents the
levels in pooled blood of two to five animals collected at different
TVs. PTHrP vs. calcium: r = 0.971;
P < 0.001; PTHrP vs. phosphate:
r = 0.962; P < 0.001.
|
|
IL-6 and PTHrP mRNA expression
The expression of hIL-6 mRNA in the three tumors paralleled the
measured values of the cytokine in blood. It was highest in NC-65 and
hardly detectable in RC-9 tumors (Fig. 2). The amount of
IL-6 mRNA increased with TV in both NC-65 and RC-8.
View larger version (36K):
[in this window]
[in a new window]
|
Figure 2. Northern blots of IL-6 mRNA expression in RC-9,
RC-8, and NC-65 tumors implanted into nude mice at different TVs.
IL-1-stimulated human fibroblasts were used as positive controls (PC)
for IL-6 mRNA expression. The RNA size marker is 18S rRNA
(arrow). 28S rRNA was analyzed as an internal
standard.
|
|
Both RC-8 and RC-9 expressed PTHrP mRNA, but there were differences
between them (Fig. 3). PTHrP mRNA expression in RC-8 was
already high at a small TV, whereas in RC-9, the expression increased
with increasing TV. No PTHrP mRNA expression was detected in NC-65.
Similar results were obtained with shorter exposure time of the films.
The difference in PTHrP mRNA size between control and experimental
tumor lines can be attributed to alternative splicing (22), as the
former originated from a squamous cell carcinoma. With use of the more
sensitive RT-PCR, IL-6 mRNA expression by RC-8 tumor cells was
visualized more clearly, also showing the increase in IL-6 mRNA with
TV, in contrast to the expression of PTHrP, which was high from the
beginning and remained constant during tumor growth (Fig. 4).
View larger version (45K):
[in this window]
[in a new window]
|
Figure 3. Northern blots of PTHrP mRNA expression in RC-9,
RC-8, and NC-65 tumors in nude mice at different TVs. SCC-4 cells
served as positive controls for PTHrP mRNA expression. RNA size markers
are 28S (upper arrow) and 18S (lower
arrow) rRNAs. 28S rRNA was analyzed as an internal standard.
|
|
View larger version (60K):
[in this window]
[in a new window]
|
Figure 4. RT-PCR analysis of IL-6, PTHrP, and ß-actin gene
expression in RC-8 tumor implanted into nude mice at different TVs. PCR
products were detected by ethidium bromide staining of 2% agarose gels
after 25 (ß-actin) or 35 (PTHrP and IL-6) cycles of amplification.
|
|
Bone histomorphometry
The BV/TV decreased exponentially with increasing TV in the
hypercalcemic RC-8 (P = 0.013) and RC-9
(P = 0.001) tumor-bearing animals, but not in NC-65
tumor-bearing animals (Fig. 5). As with hypercalcemia,
low values for BV/TV were obtained at lower TVs of RC-8 than RC-9. The
progressive decrease in BV/TV was associated with increasing plasma
PTHrP concentrations (r = 0.949; P < 0.001; Fig. 6). No such relation was found for serum IL-6
values.
View larger version (14K):
[in this window]
[in a new window]
|
Figure 6. Relation between plasma PTHrP concentrations and
BV/TV in RC-8 and RC-9 tumor-bearing mice. For plasma PTHrP, values
from pooled blood from two to five animals are shown. For BV/TV, values
represent the mean ± SD from two to five animals.
r = 0.880; P = 0.009.
|
|
Effects of anti-IL-6 treatment
A single injection of anti-IL-6 to RC-8-bearing animals
significantly decreased serum calcium concentrations. These were
2.8 ± 0.4 mmol/liter compared to 4.2 ± 0.3 mmol/liter in
vehicle-treated mice (P < 0.001; Table 2). The serum calcium concentrations after anti-IL-6
treatment did not differ from those in nontumor-bearing control animals
(2.4 ± 0.1 mmol/liter; n = 10). The decrease in serum
calcium concentrations induced by anti-IL-6 treatment was associated
with undetectable levels of PTHrP; this was 3.4 pmol/liter in the
vehicle-treated animals. There was no change in expression of IL-6 mRNA
or PTHrP mRNA with treatment, but anti-IL-6 treatment significantly
decreased the rate of growth of RC-8 tumors from 27 ± 2 to
15 ± 2 mm3/day (P < 0.001; Table 2).
In this set of experiments, no hypercalcemia developed in mice
implanted with RC-9 tumors with or without anti-IL-6 despite TV up to
500 mm3. Also, no circulating plasma PTHrP was detectable.
Treatment also had no effect on the rate of growth of RC-9 tumors.
Anti-IL-6 treatment did not influence the growth of NC-65 tumors.
View this table:
[in this window]
[in a new window]
|
Table 2. Nude mice implanted with RC-8, RC-9, and NC-65 tumor
received saline solution (controls) or anti-IL-6 as a single ip
injection at a tumor volume of 100 mm3
|
|
|
Discussion
|
|---|
Previous studies have suggested that IL-6 may contribute to the
development of malignancy-associated hypercalcemia, but its precise
role has not been determined (23). Because hypercalcemia is a known
complication of renal cell carcinomas that have also been shown to
produce IL-6 (6, 7, 8), in the present study we examined the role of IL-6
in the induction of hypercalcemia in nude mice implanted with different
human renal cell carcinoma tumor lines.
After implantation, all three tumors investigated produced significant
amounts of hIL-6. However, hypercalcemia developed only in the mice
implanted with the RC-8 and RC-9 tumors, and it was associated with
marked decreases in BV/TV. These findings suggest that under these
conditions, increased bone resorption was the primary mechanism for the
increase in serum calcium concentrations. In contrast, in the NC-65
tumor-bearing animals with the highest levels of circulating IL-6, both
serum calcium concentrations and BV/TV did not change. These results
strongly suggest that in renal cell carcinomas, IL-6 alone is not a
hypercalcemic factor and does not stimulate bone resorption. This is in
agreement with a recent study showing that IL-6 is not a powerful
bone-resorbing factor in mice in vivo, but may act
synergistically with other humoral factors in the induction of
hypercalcemia (10). PTHrP has been established as a major humoral
factor in malignancy-associated hypercalcemia, particularly that
accompanying solid tumors (1). Increased levels of IL-6 and PTHrP have
been found in hypercalcemic animals and humans, and De La Mata et
al. recently suggested that IL-6 may potentiate the action of
PTHrP on bone resorption (10). These investigators reported in studies
with nude mice inoculated with Chinese hamster ovarian cells
transfected with cDNAs for IL-6 and PTHrP that IL-6 increases the pool
of early osteoclast precursors that can further differentiate to mature
resorbing osteoclasts by the action of PTHrP. This is in line with
earlier observations of our group of the decreased effectiveness of
PTHrP on osteoclastic resorption in fetal bone explants from
IL-6-deficient mice, which could be restored with IL-6 treatment (24).
In the present study the animals that developed hypercalcemia also
produced high amounts of PTHrP, and there was an almost linear
relationship between serum calcium and phosphate concentrations and
plasma PTHrP levels. This relationship was independent of the
prevailing IL-6 levels, and for a given PTHrP value, mice with
relatively low or high IL-6 levels showed similar increments in serum
calcium concentrations. This suggests that in renal cell carcinomas,
PTHrP is the major hypercalcemic factor, and IL-6 does not appear to
have any modulatory action, a conclusion in line with a recent study of
the expression of IL-6 and PTHrP mRNAs in tumors from patients with
renal cell carcinomas (25). In this human study we found that all renal
cell carcinomas expressed IL-6 mRNA, but hypercalcemia developed only
in a patient who showed simultaneous expression of IL-6 and PTHrP mRNA.
This contrasts with the finding in squamous cell carcinomas, where all
tumors expressed PTHrP mRNA, but not every patient became
hypercalcemic. We obtained similar results in nude mice implanted with
squamous cell carcinomas (unpublished observations). The different
behavior of renal cell carcinomas was thus confirmed here by showing a
close relation between PTHrP mRNA expression and circulating levels of
PTHrP, which were independent of the expression or the circulating
values of IL-6. A synergistic effect of the two factors cannot,
however, be totally ruled out, as the concentration of IL-6 required to
trigger an increase in the osteoclast precursor pool in vivo
is not yet known. If this is low, then the present results could be
consistent with a synergistic effect of IL-6 and PTHrP in the induction
of bone resorption and consequently of hypercalcemia. We did not have a
renal carcinoma cell line producing only PTHrP to test this
possibility, but the studies of treatment of mice with an antibody to
IL-6 have been helpful in this respect.
In the present and in a previous study (11) we found that treatment of
mice implanted with the RC-8 tumor with an antibody against hIL-6
significantly reduced the rate of growth of the tumor and prevented the
increase in serum calcium concentrations. In addition, this treatment
prevented any rise in circulating PTHrP, which remained undetectable.
We have no good explanation for the lack of an antitumor effect of
anti-IL-6 in the other tumors. The dose of antibody used has been found
to effectively neutralize values of circulating hIL-6 similar to those
obtained in our experiment (Dr. L. Aarden, personal communication).
Furthermore, the immunoassayable IL-6 in the mice of the present study
was bioactive, as confirmed by measurement of some samples in the mouse
hybridoma B9 bioassay for IL-6 (own unpublished observations).
IL-6 has been previously recognized as an autocrine growth factor in
some renal cell carcinomas (26). It may, therefore, be that in these
carcinomas a synergism between IL-6 and PTHrP in the induction of
hypercalcemia does not occur at the level of osteoclastogenesis, but
is, rather, the result of the stimulating action of IL-6 on tumor
growth. In cancer cells that have the capacity to produce PTHrP, IL-6,
by stimulating their growth, enhances PTHrP production, which, in turn,
stimulates bone resorption and induces hypercalcemia. Treatment with an
antibody against IL-6 disrupts this sequence of events and prevents the
increase in serum calcium concentrations. These observations in mice
may also have practical clinical implications. Patients with renal cell
carcinomas and PTHrP-induced hypercalcemia initially respond favorably
to treatment with antiresorptive agents, such as the bisphosphonates.
Hypercalcemia, however, recurs and then it is difficult to control,
probably because of the increased renal tubular reabsorption of calcium
induced by PTHrP, an action that cannot be blocked by antiresorptive
agents. In this setting, anti-IL-6 antibodies may theoretically offer a
better therapeutic alternative. However, this possibility needs to be
tested in humans in vivo.
An interesting additional observation in our studies was that the mice
implanted with NC-65 tumors and having the highest levels of IL-6 did
not develop cachexia. In contrast, in the other mice there was a
significant weight loss. IL-6 has been implicated in the cachexia of
malignant tumors, either alone or in association with other cytokines,
such as IL-1 and tumor necrosis factor (27). The results of the present
study strongly suggest that at least in renal cell carcinomas, IL-6
alone cannot be considered as a factor inducing cachexia, and further
studies in this direction are warranted.
Our findings, therefore, demonstrate that IL-6 is not directly involved
in the induction of hypercalcemia in renal cell carcinomas and that
PTHrP is the main humoral factor responsible for this effect, primarily
through stimulation of bone resorption. IL-6 may, however, play an
important role by stimulating the growth of renal tumors that have the
capacity to produce PTHrP.
Received September 13, 1996.
|
References
|
|---|
-
Mundy GR 1990 Pathophysiology of
cancer-associated hypercalcemia. Semin Oncol 17:10–15[Medline]
-
Martin TJ 1990 Properties of parathyroid
hormone-related protein and its role in malignant hypercalcemia. Q
J Med 76:771–786[Free Full Text]
-
Löwik CWGM, van der Pluijm G, Bloys H, Hoekman K,
Bijvoet OLM, Aarden LA, Papapoulos SE 1989 Parathyroid hormone
(PTH) and PTH-like protein (PLP) stimulate interleukin-6 production by
osteogenic cells: a possible role of interleukin-6 in
osteoclastogenesis. Biochem Biophys Res Commun 162:1546–1552[CrossRef][Medline]
-
Ishimi Y, Miyaura C, Jin CH, Akatsu T, Abe E, Nakamura
Y, Yamaguchi A, Yoshiki S, Matsuda T, Hirano T, Kishimoto T, Suda
T 1990 IL-6 is produced by osteoblasts and induces bone
resorption. J Immunol 145:3297–3303[Abstract]
-
Jilka R, Hangoc G, Girasole G, Passeri G, Williams DC,
Abrams JS, Boyce B, Broxmeyer H, Manolagas SC 1992 Increased
osteoclast development after estrogen loss: mediation by interleukin-6.
Science 257:88–91[Abstract/Free Full Text]
-
Koo AS, Armstrong C, Bochner B, Shimabukuro T, Tso CL,
deKernion JB, Belldegrun A 1992 Interleukin-6 and renal cancer:
production, regulation and growth effects. Cancer Immunol Immunother 35:97–105[CrossRef][Medline]
-
Takenawa J, Kaneko Y, Fukumoto M, Fukatsu A, Hirano T,
Fukuyama H, Nakayama H, Fujita J, Yoshida O 1991 Enhanced
expression of interleukin-6 in primary human renal cell carcinomas.
J Natl Cancer Inst 83:1668–1672[Abstract/Free Full Text]
-
Tsukamoto T, Kumamoto Y, Miyao N, Masumori N, Takahashi
A, Yanase M 1992 Interleukin-6 in renal cell carcinoma. J
Urol 148:1778–1782[Medline]
-
Fukumoto S, Matsumoto T, Harade S-I, Fujisaki J, Kawano
M, Ogata E 1991 Pheochromocytoma with pyrexia and marked
inflammatory signs: a paraneoplastic syndrome with possible relation to
interleukin-6 production. J Clin Endocrinol Metab 73:877–881[Abstract/Free Full Text]
-
De La Mata J, Uy HL, Guise TA, Story B, Boyce BF, Mundy
GR, Roodman GD 1995 Interleukin-6 enhances hypercalcemia and bone
resorption mediated by parathyroid hormone-related protein in
vivo. J Clin Invest 95:2846–2852
-
Weissglas M, Schamhart D, Löwik C, Papapoulos S,
Vos P, Kurth K-H 1995 Hypercalcemia and cosecretion of
interleukin-6 and parathyroid hormone-related peptide by a human renal
cell carcinoma implanted into nude mice. J Urol 153:854–857[CrossRef][Medline]
-
Otto U, Huland H, Baisch H, Klöppel G 1985 Transplantation of human renal cell carcinoma into NMRI nu/nu mice.
Effect of irradiation on tumor acceptance and tumor growth. J Urol 134:170–174[Medline]
-
Höhn W, Schröder FH 1978 Renal cell
carcinoma: two new cell lines and a serially transplantable nude mouse
tumor (NC65). Invest Urol 16:106–112[Medline]
-
Kurth KH, Van Dongen JW, Romijn JC, Lieber MM,
Schröder FH 1994 Assay evaluability of drug testing systems
determined with human renal carcinoma cell lines. World J Urol 2:146–155
-
Kurth KH, Weissglas MG, Romijn JC, Schröder FH,
Van Dongen JW 1983 Chemotherapy of human kidney carcinoma in
vitro and in vivo using the nude mouse model. Akt Urol 14:223–229
-
Brakenhoff JPJ, Hart M, De Groot ER, Di Padova F, Aarden
LA 1990 Structure-function analysis of human IL-6. Epitope mapping
of neutralizing antibodies with amino- and carboxyl-terminal deletion
mutants. J Immunol 145:561–568[Abstract]
-
Kaashoek JGJ, Mout R, Falkenburg IHF, Willemze R, Fibbe
W, Landegent IE 1991 Cytokine production by the bladder carcinoma
cell line 5637: rapid analysis of mRNA expression levels using a
cDNA-PCR procedure. Lymphokine Cytokine Res 10:231–235[Medline]
-
Boxman I, Löwik C, Aarden L, Ponec M 1993 Modulation of IL-6 production and IL-1 activity by
keratinocyte-fibroblast interaction. J Invest Dermatol 101:316–324[CrossRef][Medline]
-
Parfitt AM, Drezner MK, Glorieux FH, Kanis JA, Malluche
H, Meunier PJ, Ott SM, Recker RR 1987 Bone histomorphometry:
standardization of nomenclature, symbols and units. J Bone Miner
Res 2:595–610[Medline]
-
Duncan DB 1955 Multiple range and multiple F tests.
Biometrics 11:1–42
-
Kramer CY 1956 Extension of multiple range tests to
groups means with unequal numbers of replications. Biometrics 12:307–310[CrossRef]
-
Ikeda K, Mangin M, Dreyer BE, Webb AC, Posillico JT,
Stewart AF, Bander NH, Weir EC, Insogna KL, Broadus AE 1988 Identification of transcripts encoding a parathyroid hormone-like
peptide in messenger RNAs from a variety of human and animal tumors
associated with humoral hypercalcemia of malignancy. J Clin Invest 81:2010–2014
-
Löwik CWGM 1992 Differentiation inducing
factors: leukemia inhibiting factor and interleukin-6. In: Gowen M (ed)
Cytokines and Bone Metabolism. CRC Press, Boca Raton, pp 300–323
-
Most W, van Beek E, Ruwhof C, van Bezooyen R, Ederveen
A, Kopf M, Papapoulos S, Löwik C 1994 Osteoclastic
resorption in interleukin-6 deficient mice. J Bone Miner Res
[Suppl 1] 9:S132 (Abstract)
-
Schweitzer DH, Boxman ILA, Löwik CWGM, van Krieken
JHJM, Weissglas MG, Baatenburg de Jong RJ, Papapoulos SE 1995 Parathyroid hormone related protein and interleukin-6 mRNA expression
in larynx and renal cell carcinomas from normocalcaemic and
hypercalcaemic patients. J Clin Pathol 48:896–900[Abstract/Free Full Text]
-
Miki S, Iwano M, Miki Y, Yamamoto M, Tang B, Yokokawa K,
Sonoda T, Hirano T, Kishimoto T 1989 Interleukin-6 (IL-6)
functions as an in vitro autocrine growth factor in renal
cell carcinomas. FEBS Lett 250:607–610[CrossRef][Medline]
-
Greenberg AS, Nordan RP, McIntosh J, Calvo JC, Scow RO,
Jablons D 1992 Interleukin-6 reduces lipoprotein lipase activity
in adipose tissue of mice in vivo and in #T3–L1 adipocytes:
a possible role for interleukin-6 in cancer cachexia. Cancer Res 52:4113–4116[Abstract/Free Full Text]
This article has been cited by other articles:
|
|
|
|
 
H. Brady, M. Doubleday, L. M. Gayo-Fung, M. Hickman, S. Khammungkhune, A. Kois, S. Lipps, S. Pierce, N. Richard, G. Shevlin, et al.
Differential Response of Estrogen Receptors alpha and beta to SP500263, a Novel Potent Selective Estrogen Receptor Modulator
Mol. Pharmacol.,
March 1, 2002;
61(3):
562 - 568.
[Abstract]
[Full Text]
[PDF]
|
|
|
Top
Abstract
Introduction
, NC-65 (r = 0.004;
P = 0.988).