[CANCER RESEARCH 43, 3763-3769,
August 1983]
Characteristics of a Calcitonin-responsive
Human Osteosarcoma1
Cell Line Derived from a
Gabriel Eilon, Janice Perkins, and Michael V. Viola2
Departments of Medicine [G. £.,M. V. V.] and Pediatrics [J. P.], The University of Connecticut School of Medicine, Farmington, Connecticut 06032
ABSTRACT
Although the primary cell type in human osteosarcoma is
usually a neoplastic osteoblast, numerous other mesenchymal
cell types may coexist in the same tumor. Previously described
cloned, long-term osteosarcoma cell lines have had an osteoblastic phenotype. In this report, we describe a nonosteoblastic,
long-term cell line derived from an osteosarcoma in a patient
with Paget's disease. The cell line (FM-2) is nontransformed in
having a low saturation
density
and anchorage-dependent
growth, and it is nontumorigenic in nude mice. Important features
of its fine structure include numerous elongated mitochondria,
abundant Golgi and lysosomes, and a poorly developed rough
endoplasmic reticulum. The line has high levels of lysosomal
enzymes (acid phosphatase and A/-acetylglucosaminidase) and
low levels of alkaline phosphatase. It lacks numerous macro
phage markers (lysozyme, C3, Fc receptors, and M1 antigen).
The FM-2 line had a dose-dependent cyclic AMP response (7fold increase) following treatment with calcitonin but not with
parathormone. In 125l-calcitonin-binding experiments, we calcu
lated ~5.3 ±0.2 x 103 receptor sites/cell with a K, of 1.8 ±0.1
x 10~9 M. Conditioned medium from the FM-2 line was a potent
stimulator of calcium release as assayed in a 45Ca-labeled fetal
rat bone organ culture. This activity was not prostaglandin,
vitamin D, parathormone, or epidermal growth factor, which are
known stimulators of bone résorption.The FM-2 line does not
appear to be derived from an osteoblast, macrophage, or fibroblast and may represent a calcitonin-responsive bone stem cell.
INTRODUCTION
There are a number of descriptions of continuous cell lines
from osteosarcomas of animal (8,16, 17) and human (9,19, 31,
34) origin. Many of these cell lines have been well-characterized
with respect to their morphology, karyotype, and properties
indicating degree of neoplastic transformation (;'.e., saturation
density, anchorage independence, and tumorigenicity in immunecompromised experimental animals). However, more complete
descriptions of the phenotype of cell lines from osteosarcomas
have been reported less frequently. This information is of impor
tance, since numerous pathological studies have emphasized
the heterogeneity of cell types comprising this tumor. For ex
ample, human osteosarcomas are usually comprised of boneforming cells (osteoblasts) in various stages of differentiation,
but other areas of the tumor may contain primitive nonidentifiable
cells, fibrosarcoma cells, myofibroblasts, chondroblasts, and
multinucleated cells resembling osteoclasts (27, 40).
' Supported by Grant CA 27792 from the National Cancer Institute.
2To whom requests for reprints should be addressed.
Received September 2, 1982; accepted April 29, 1983.
The most completely characterized osteosarcoma cell lines
are of rat and human origin and have an osteoblastic phenotype
(16, 17, 34). The distinguishing features of these lines include
parathyroid hormone-responsive adenylate cyclase, elevated al
kaline phosphatase, synthesis of X-carboxyglutamic acid-con
taining bone protein, and production of mineralized tumors in
inoculated rats. In this paper, we report the phenotypic charac
teristics of a cloned cell line (FM-2) derived from a human
osteosarcoma whose fine structure, enzyme content, and calci
tonin responsiveness are unique among cell lines derived from
this tumor type. Furthermore, the cells secrete, into the super
natant medium, a potent stimulator of calcium release as assayed
in fetal rat bone organ cultures.
MATERIALS
AND
METHODS
Establishment of the FM-2 Cell Line. This Å“il line was derived from
tumor tissue obtained from an open femoral biopsy of a patient with
osteosarcoma. The patient had Paget's disease of bone also. The tumor
specimen was minced into small fragments and treated with trypsin.
Single cells in the supernatant fluid were seeded onto plastic Retri dishes.
The cell line was propagated in Dulbecco's minimum essential medium
with 10% newborn calf serum and has been in continuous culture for 2
years. The FM-2 clone was established by single-cell cloning in microtiter
wells and has been in continuous culture for 18 months. The agar
suspension culture technique on FM-2 cells was as described by Mc
Allister ef al. (19).
Electron Microscopy. Cultured cells were fixed in situ in Retri dishes
with 4% glutaraldehyde in 0.1 M sodium cacodylate, pH 7.0, at room
temperature for 1 hr followed by postfixation in osmium tetroxide in 5
HIM Veronal buffer. Cells were then dehydrated in alcohol and embedded
in Epon. Small pieces of Epon containing embedded cells were removed,
and thin sections were cut perpendicular to the surface of the plate. Thin
sections were stained with uranyl acetate and lead citrate and examined
with a Hitachi HU-11E-1 electron microscope. For scanning electron
microscopy, cells were fixed and dehydrated as above and then critical
point dried with CO2. Cells were mounted on stubs, coated with goldpalladium (—300 A thickness), and examined with a Hitachi H-300 with
an H-3010 scanning attachment at 20 kV.
Biochemical Studies. Enzyme assays were performed on cell lysates
(in 1% Triton X-100) using standard methodology for the following
enzymes: alkaline phosphatase (14), acid phosphatase and N-acetylglucosaminidase (2), 5'-nucleotidase (4), aminopeptidase (37), and lysozyme
(22). Collagen synthesis was measured by incorporation of [3H]proline
into collagenase-digestible
protein in cells using the method of Peterkof-
sky (24). C3 and Fc receptors and M1 antigen were assayed using
standard methods with commercially prepared reagents from Cordis
Laboratories (Miami, Fla.) and Ortho Diagnostics, Inc. (Raritan, N. J.). In
vitro phagocytosis was tested by exposing freshly plated cells to 50
particles/cell followed by incubation at 37°for 45 min and examination
by phase-contrast microscopy. Particles tested were 1-Mm latex beads,
opsonized yeast cells, and glutaraldehyde-treated
RBC. Histochemical
identification of acid phosphatase (11 ) and alkaline phosphatase (1) was
by established methods.
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3763
G. Eilon et al.
AMP Assays. cAMP3 levels were determined on Å“il extracts (cells
extracted with 1.2 M tricarboxylic acid) using a New England Nuclear
(Boston, Mass.) kit by the method of Farmer ef al. (5). cAMP was
assayed in the presence of 5 x 10~4 M isobutylmethylxanthine.
Binding of 125l-Labeled Calcitonin to Cells. Both labeling of salmon
calcitonin with 125Iby the chloramine-T method and binding of labeled
hormone to cells were performed as described by Lamp ef al. (13).
Replicate cultures in 4.5-cm wells were exposed to 50,000 cpm of 125Icalcitonin (100 to 200 nC\/ng) in 1 ml of buffer (15 rriM Tris, pH 7.4; 120
rnw NaCI; 4 ITIM KCI; 1.6 mw MgSO4; 2 mw NaH2PO4; 10 HIM glucose;
and 0.1% human serum albumin) containing increasing amounts of
unlabeled calcitonin. After 2 hr at 37°, cells were washed 3 times with
phosphate-buffered
saline (washes saved for counting of nonbound
radioactivity) and lysed with 0.5 ml of 0.5 M NaOH, and acid-precipitable
radioactivity was counted in a ^-counter.
Bone Resorption Assay. Supernatant fluids from a 2-day culture were
mixed in equal volume with fresh medium and placed on fetal rat radius
and ulna cultures labeled with 4>JCaand assayed as described previously
(25).
RESULTS
Histology and Ultrastructure of Original Osteosarcoma. The
tumor specimen was obtained from an adult male with biopsyproven Paget's disease of bone. He had not received previous
therapy for the latter disease. The tumor arose in the femoral
head and by light microscopy was comprised of large anaplastic
cells with pleomorphic, hyperchromatic nuclei. In some areas,
sheets of tumor cells were seen with no intervening stroma; in
other areas, typical osteoid matrix was seen between clumps of
tumor cells. Examination of the tumor specimen by transmission
electron microscopy revealed 3 distinct cell types. Typical malig
nant osteoblasts were the most abundant cell type. These were
rounded cells with eccentrically placed nuclei and cytoplasm
containing abundant dilated rough endoplasmic reticulum. A
second frequent cell type were large, undifferentiated cells con
taining markedly irregular nuclei with scant cytoplasm containing
few organelles. These cells were similar to the "primitive" cells
described in human osteosarcomas by Reddick ef al. (27). A
third cell type seen less frequently than osteoblasts and primitive
cells was spindle shaped and contained a centrally placed nu
cleus and small amounts of rough endoplasmic reticulum. These
cells contained bundles of 60-Àfilaments with dense bodies and
were tentatively identified as myofibroblasts. In summary, both
light- and electron-microscopic studies confirmed the diagnosis
of an osteoblastic osteosarcoma.
Growth Characteristics and Karyotype. The FM-2 cell line is
a monolayer culture that is contact-inhibited in minimum essential
medium with 10% calf serum and exhibits no areas of "piling up"
of cells. The cell line does not form colonies in semisolid agar
(both early and late passages tested) and is nontumorigenic
when inoculated into nude mice (largest inoculum, 5 x 106 cells).
The doubling time of the cell line is 23 hr, and it has a saturation
density of 1.5 x 104 cells/sq cm.
Two separate cytogenetic studies were performed on the FM2 cell line. The first was done after the cloned line had been in
continuous culture for 4 months. Thirty-seven well-spread Gbanded metaphases were counted. The chromosome count was
hypotetraploid, ranging in number from 71 to 87, with a mode of
3 The abbreviation used is: cAMP, cyclic adenosine 3':5'-monophosphate.
3764
81. Chromosomes from 20 cells were examined in detail. Chro
mosomes 3, 6, 7, 8, 9, 11, and 19 were usually found as
tetraploid, and chromosomes 1, 2, 4, 5, 10, 13, 14, 17, 18, 20,
21, and 22 were usually found as triploid. Approximately 10 to
17 marker chromosomes (including unidentifiable translocations,
acentric fragments, and minutes) could be found in each chro
mosome spread. Of these marker chromosomes, 5 could be
consistently identified in 100% of the cells and were predomi
nantly C-group in size or larger. An additional 5 marker chro
mosomes could be seen in approximately 30% of the cells and
represented chromosomes of variable size. A similar analysis
was performed after 1 year of continuous culture of the FM-2
line and yielded a similar cytogenetic pattern. Thus, this cell line
is characterized by a single mode of hypotetraploid karyotype
and was stable over the period of observation.
Fine Structure. The cell line is composed of large, flattened
cells, whose dorsal surface contains numerous microprojections
(Figs. 1 and 2A). These include microvilli, retraction microfibrils,
and numerous blebs and folds over the nucleus. These structures
are not unique for a specific cell type and have been observed
on the surfaces of macrophages, epithelioid cells, and fibroblasts,
particularly when in long-term culture (summarized in Ref. 35).
The nuclei are large, with a single prominent nucleolus and
marginated chromatin. Intranuclear virus-like fibrillary structures,
which have been seen in osteoclasts of some patients with
Paget's disease, were not observed (20). Prominent cytoplasmic
structures included a well-developed Golgi zone with numerous
adjacent lysosomes, large numbers of elongated mitochondria,
and clusters of ribosomes. Numerous vacuoles, particularly autophagocytic vacuoles, were evident. In early passages, abun
dant, dense membrane-bound granules of varying sizes were
seen, but, in later passages, fewer granules were evident. The
prominent rough endoplasmic reticulum of osteoblasts and cy
toplasmic glycogen lakes of preosteoblasts were absent (30).
Biochemical Assays. A number of biochemical phenotypic
markers were assayed to identify the cell of origin of the FM-2
cell line (summarized in Table 1). We compared it to a rat
osteoblast-like cell line (ROS 17/2) (16) and to a murine macro
phage cell line (J774) (32) which expresses a number of consti
tutive and induced macrophage products. The osteoblastic prop
erties of the ROS 17/2 cell line include high levels of alkaline
phosphatase and responsiveness to parathormone. However, it
is a malignant sarcoma and expresses a number of enzymes
(e.g., lysosomal enzymes) in excess of that found in osteoblasts
in vivo (26). We compared the FM-2 cell line to a macrophage
line, because there is considerable indirect evidence that osteo
blasts are derived from cells of the macrophage-monocyte series
(38).
The FM-2 cell differed from ROS 17/2 in having a low level of
alkaline phosphatase, an enzyme whose presence in bone cells
is characteristic of osteoblasts. The levels of lysosomal enzymes
(acid phosphatase and /v-acetylglucosaminidase) assayed were
comparable in the FM-2 cell line and the murine macrophage. In
histochemical staining experiments, all FM-2 cells stained heavily
for acid phosphatase (Fig. 1B), and none stained for alkaline
phosphatase, indicating that the FM-2 line is composed of a
relatively homogeneous populations of cells. The pattern of
ectoenzymes was similar in the FM-2 cell and J774 (i.e., high 5'nucleotidase and low aminopeptidase) and is characteristic of
activated macrophages. The elevated levels of both ectoen
zymes in the ROS 17/2 cell line are unexplained. Although the
CANCER
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Osteosarcoma Cell Line
Table 1
Comparison of FM-2, rat osteoblastic osteosarcoma (ROS 17/2), and mouse macrophage (J774) cell lines
Enzyme assays were performed as described in "Materials and Methods" on lysates from ~5 x 105 cells from each cell line. M1 antigen is of human origin; therefore,
ROS 17/2 and J774 were not tested. Ninety-five % of cells from a primary human macrophage culture were positive in an indirect immunofluorescence
antibody to M1 antigen. Phagocytosis assays were performed in duplicate on monolayer cultures (60-mm Petri plates) and examined by phase microscopy.
markersCell
linesFM-2
Osteoblastic
phosphatase80.40
±0.03d' e
ROS 17/2
J774Alkaline
18.6
0.9
enzymesAcid
phossynthesis61.42 phatase"1.70
±0.05°
±1.90
4.8 ±0.30
1.4
±0.10Collagen NDLysosomal
2.1
assay with
markersM1
glucosaminidasea7.10
±0.30e
±0.05e
Negative<1
+ 0.03
±0.01
T912.0
±0.02
5.8 ±0.20
198.0 ±0.12
6.5 ±0.70
±0.20W-acetyl4.0 ±0.20Ectoenzymes5'-Nucleotidase82.60
4.3 ±0.40Amino0.7 ±0.40Macrophage
±0.05
nmol/mg protein/hr.
" Percentage of [3H]proline incorporated into collagenase-digestible
c Enzyme activity in 1 Mg egg lysozyme (5 x 10s cells assayed).
0 Mean ±S.E. of 4 determinations.
e Significantly different from value of ROS 17/2 (p < 0.01, f test).
' Significantly different from value of J774 (p < 0.05, ( test).
9 T, not tested.
anti-
peptidase80.60 gen<l'
Lysozyme0
TC3
and Fc
receptorsNegative
tosisNegative
Negative
Negative
PositivePhagocy
Positive
protein.
FM-2 cell line has a number of properties characteristic of acti
vated macrophages, it lacks lysozyme (a constitutive enzyme of
most macrophages), surface C3, Fc receptors, and a surface
antigen (M1) relatively specific for human macrophages and
lymphoid cells (3). In addition, it is nonphagocytic.
Response of FM-2 Cell Line to Calcitonin. The cAMP re
sponse was measured in FM-2 cells and ROS 17/2 cells after
treatment with either calcitonin or parathormone (Chart 1). A 7fold increase in cAMP levels was observed in the FM-2 cell line
following calcitonin treatment. A minimal response was noted
after parathormone treatment. The reverse of this response was
seen after hormone treatment of the ROS 17/2 cell line. The
ROS 17/2 cell line has been shown previously to respond pref
erentially to parathormone. Both cell lines had a maximal cyclic
AMP response to the appropriate hormone 4 to 8 min after
initiation of treatment. There was a dose-dependent response of
cAMP to increasing concentrations of calcitonin. The maximum
cAMP response in 5 min was obtained with calcitonin concentra
tions above 2 x 10~10M (data not shown).
FM-2
CT
8070605040O>
30203
E 10Q.
PTH
ROS \7/2
<
U
The binding of 125l-labeledcalcitonin to intact FM-2 cells in the
presence of increasing concentrations of unlabeled hormone is
shown in Chart 2A. These experiments were performed in quad
ruplicate at 37°for 2 hr. The long incubation time is necessary
because of the slow rate of dissociation of calcitonin from its
receptor in other cell systems (6, 13), necessitating long incu
bation periods to establish a steady state of labeled hormone in
the "bound" and "free" fractions. Nonspecific binding of calcitonin
(i.e., binding in the presence of 1 ^M unlabeled calcitonin) was
less than 5% of the total input radioactivity.
Scatchard plot analysis of the data obtained in competition
experiments yielded a straight-line plot (Chart 2B), suggesting
the existence of a single class of binding sites for calcitonin on
FM-2 cells. The dissociation constant for the intact cell receptor
was approximately 1.8 ±0.1 x 10~9 M, and the receptor number
per cell was 5.3 ±0.2 x 103. In terms of receptor abundance,
the FM-2 cell line is comparable to the breast cancer cell line,
MCF7 (~4,700 receptor sites/cell), has considerably more than
lymphocyte cell lines (~300), but fewer than the BEN lung
carcinoma line (-30,000) (6, 7, 18).
Bone Resorption Studies. The effects of dilutions of FM-2
conditioned medium on 45Ca release from fetal rat bone were
measured (Chart 3). Low levels of stimulation were observed in
undiluted and 1:2 dilutions of conditioned medium. Optimum
Minutes
Chart 1. cAMP levels (pmol/mg protein) in FM-2 cell line and ROS 17/2 cell line
following hormone treatment. Cells (5 x 105) were treated with human parathor
mone 1-84 (200 ng/ml) (PTH) or salmon calcitonin (50 milliunits/ml) (CT), and cells
were harvested at the time specified. Bars, S.D.
activity was seen at dilutions of 1:5, and activity was still present
in dilutions of 1:20. The effect of conditioned medium from other
cell lines on 45Ca release from fetal bone organ cultures was
studied also (Table 2). Medium from a mouse macrophage line
(J774) was shown to stimulate 45Carelease; however, growth of
J774 cells in the presence of ¡ndomethacin reduced this effect
by 60%. Indomethacin (and ibuprofen) inhibit(s) synthesis of
prostaglandins, and it has been shown that prostaglandins stim
ulate bone résorptionin bone organ cultures (12). Ibuprofen and
indomethacin have no effect on 45Carelease when added directly
to bone organ cultures (data not shown). Bone résorptionstim
ulation of FM-2 conditioned medium was unaffected by growth
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3765
G. Eilon et al.
composed primarily of neoplastic osteoblasts (in varying stages
of differentiation) and undifferentiated cells which could not be
classified as a specific cell type. The tumor developed in Pagetic
bone, and we have previously reported virus-like nuclear inclu
o
sions in undifferentiated cells in this tumor (36). These fibrillar
inclusions resembled the nuclear inclusions found in osteoclasts
in Paget's disease (20). We have never observed similar inclu
o
m
sions in the FM-2 cell line or its parental line despite frequent
examination of the cell lines throughout their passage in culture.
The FM-2 cell line exhibited a number of characteristics of
transformed cells, including ease of establishment in culture and
immortalization in vitro. However, using the more stringent cri
teria of attachment-independent
growth and tumorigenicity in
nude mice, it has a nontransformed phenotype. There are a
number of cell lines derived from neoplastic tumors, including
osteosarcoma (28), which express only a partially transformed
phenotype in vitro.
We have attempted to identify the cell of origin of the FM-2
n
CM
20
SCI (nM)
0020
.
B
u
ÃœJ
(E
LL
0015
ODIO
CD
0005
IO
20
30
40
50
SCI BOUND (pM)
Chart 2. A, binding of 125l-labeled salmon calcitonin (SCT) to FM-2 cells and
competition with unlabeled salmon calcitonin; 5 x 10s cpm of labeled calcitonin
was used at a spécifieactivity of 150 iiCi/^g: B, Scatchard analysis of binding data.
60
50
e
O
40
*
30
I-
0
U
O.
clone by its fine structure, enzyme content, and response to
hormones. The tumor specimen from which it was derived con
tained not only the neoplastic cells described above but sparse,
normal stromal cells and vascular elements. Furthermore, we are
aware that there can be phenotypic drift or inappropriate expres
sion of certain cell markers in long-term culture which may make
identification of the cell of origin ambiguous. The osteosarcoma
cell lines, whose phenotypes have been described previously,
have had osteoblastic features. In contrast, the FM-2 cell line
was nonosteoblastic in having a poorly developed rough endoplasmic reticulum and low alkaline phosphatase; in addition, it
was nonresponsive to parathormone. The fine structure of the
cytoplasmic contents bears some resemblance to osteoclasts
(10) and putative preosteoclasts (29), particularly the abundant
elongated mitochondria, prominent Golgi zones, and numerous
lysosomes. Furthermore, calcitonin responsiveness has been
proposed as an indication that a bone cell is in the osteoclast
series (15, 23). We have found that the FM-2 cell line possesses
high-affinity receptors for calcitonin and a calcitonin-induced
cAMP response. However, in addition to osteoclasts (39), calci
tonin receptors have been detected on kidney cells (39), lymphoid
cell lines (18), human breast cancer cell lines (7), and on a human
Table 2
Effect of cell line supernatants on KCa release from bone organ cultures
20
Supernatant fluids from a 2-day culture were mixed in equal volume with fresh
medium and placed on fetal rat radius and ulna cultures labeled with 45Ca as
IO
1=2
I--IO
l'20
CONDITIONED
MEDIA
DILUTIONS
Chart 3. Effects of FM-2 conditioned medium on 45Ca release from fetal rat
bone organ cultures. Difference percentage of 45Ca release is the difference
between the percentage of 45Ca release in test cultures minus the percentage of
45Ca release in control cultures. Assays were performed in quadruplicate; points,
described previously (25). Indomethacin and ibuprofen were added
supernatants during the 2-day incubation period. Treatment of bone
with these 2 drugs in the absence of conditioned medium had no
release. Values are expressed as W0a release per mg cell protein
to the cell line
organ cultures
effect on 45Ca
in the cell line
from which the supernatant was obtained. Percentage of calcium release equals
45Ca in the supernatant divided by total "Ca (supernatant plus bone). Difference
percentage of 45Ca release equals percentage of release in test fetal bone culture
minus percentage of release in a fetal bone culture to which no conditioned medium
has been added.
Release)FM-2
Conditions (A% of wCa
mean; bars, S.D.
of FM-2 cells in the presence of indomethacin or ibuprofen. Also,
these drugs had no effect on the growth of FM-2 or J774 cells.
DISCUSSION
The original tumor from which the FM-2 cell line was derived,
when examined histologically and by electron microscopy, was
3766
M)13.9
(10-*
±1.0°
±1.1
1.3 + 0.2
ROS 17/2
1.9 ±0.2
1.4 ±0.1
3T3
1.1 ±0.1
1.0 ±0.2
0.9 ±0.1
20.1 ±0.1fiIndomethacin
J774Control15.7±0.8a'6
8.1 ±0.1Ibuprofen(10~5M)16.1
Not tested
a Mean ±S.E. for 4 cultures in each treatment group.
0 Significantly greater than values for 3T3 and ROS 17/2 cells (p < 0.01, ( test).
c Significantly greater than values for 3T3, ROS 17/2. and J774 cells (p < 0.01,
( test).
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VOL. 43
Osfeosarcoma Cell Line
lung cancer line (6). In the lung cancer line, it is not clear if the
presence of these receptors represents the inappropriate expres
sion of a peptide hormone receptor in a neoplastic cell.
Since osteoclast cell lines do not exist, the presence of calcitonin receptors in a long-term cell line of presumed bone origin
will be useful in the study of this hormone with its "physiological"
receptor. A number of unusual aspects in the interaction of
calcitonin with its receptor have been found in the breast and
lung cancer cell lines (e.g., slow and incomplete reversal of
hormone binding and persistent activation of adenylate cyclase
after brief exposure to hormone). It will be of interest to compare
those results with results on FM-2 cells when they are studied
further.
In summary, it is difficult to unequivocally identify the cell of
origin of the FM-2 cell line. Its fine structure, enzyme content,
and calcitonin responsiveness suggest that it is not an osteoblast, macrophage, or fibroblast. It has a number of phenotypic
characteristics associated with osteoclasts; however, we have
little knowledge of the phenotype of the preosteoclast. Never
theless, the FM-2 cell may represent a bone stem cell, and we
are presently attempting to induce differentiation of this cell line
using pharmacological agents and varying culture and substrate
conditions.
It was of interest to determine whether the FM-2 cell line
elaborates soluble factors of possible importance in normal bone
physiology. We found that conditioned medium from FM-2 cells
contains a factor which stimulates 45Ca release in bone organ
cultures. Tashjian ef al. (33) have shown that conditioned medium
from a mouse fibrosarcoma line stimulates bone résorption in
organ cultures and that this effect is mediated by prostaglandin
E2. Growth of FM-2 cells in the presence of inhibitors of prosta
glandin synthesis had no effect on the presence of bone résorp
tion stimulation activity in conditioned medium. We have partially
purified this factor from FM-2 conditioned medium, and a detailed
description of its biochemical properties has been submitted for
publication.4 We found that the factor is not prostaglandin or
parathyroid hormone, as determined by radioimmunoassays.
Furthermore, it is not vitamin D, nor does it compete for epidermal
growth factor receptors (data not presented). This factor may be
similar in its biological activity to "osteoclast activating factor,"
which is a lymphokine elaborated by stimulated lymphocytes and
some neoplastic hematopoietic cell lines (21). However, this is
the first bone résorptionstimulation factor of this type derived
from a presumed bone cell and, thus, may be of importance in
normal bone physiology as well as in the pathological bone
résorptionsometimes associated with neoplastic cells.
ACKNOWLEDGMENTS
We thank Dr. Bijay Mukherji and Dr. Peter Cooke for their assistance and Dr.
Lawerence Raisz for helpful suggestions.
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CANCER
RESEARCH
VOL. 43
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3769
Characteristics of a Calcitonin-responsive Cell Line Derived
from a Human Osteosarcoma
Gabriel Eilon, Janice Perkins and Michael V. Viola
Cancer Res 1983;43:3763-3769.
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