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From the Departamento de Bioquímica y Biología
Molecular,*
and Morfología y Biología
Celular,
Facultad de Medicina, Universidad de
Oviedo, Oviedo, and the Servicio de
Cirugía,
Hospital de Jove,
Gijón, Spain; and the Department of Biochemistry and Molecular
Dentistry,§
Okayama University Dental School,
Okayama, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The wide spectrum of substrates susceptible to proteolytic degradation by collagenase-3, together with the presence of this enzyme in breast carcinomas but not in normal mammary gland nor in benign mammary lesions, has suggested that it could play a critical role in the uncontrolled lytic processes occurring during malignant tumor progression.1 In agreement with this proposal, preliminary studies on the prognostic value of collagenase-3 in breast cancer have revealed that this enzyme is a marker of poor clinical outcome in breast cancer patients (F. Vizoso, unpublished data). In addition, recent studies have shown that collagenase-3 is also overexpressed by a subset of laryngeal squamous cell carcinomas with extensive local invasion.13 However, at present, there is no information on the possible production of this enzyme by tumors other than breast carcinomas and squamous cell carcinomas.1,13-15 In this regard, the finding that collagenase-3 is produced by chondrocytes during human fetal development and in joint-destructive processes prompted us to examine the possibility that this enzyme could be also associated with tumor processes involving these cells. The malignant cartilage-forming tumors or chondrosarcomas account for approximately 25% of all malignant tumors arising from the skeletal system and are the most frequently diagnosed bone tumors in patients older than 50 years.16-21 In this work, we provide evidence that collagenase-3 is produced in significant amounts by human chondrosarcoma cells. In addition, analysis of factors potentially responsible for this effect has revealed that bFGF is a strong inducer of collagenase-3 expression in chondrosarcoma cells. Finally, we have evaluated by immunohistochemistry the presence of collagenase-3 in a series of benign and malignant cartilage-forming tumors.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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All media and supplements for cell culture were obtained from
Sigma Chemical Co. (St. Louis, MO) except for fetal calf serum, which
was from Boehringer Mannheim (Mannheim, Germany). Basic fibroblast
growth factor (bFGF), acidic FGF (aFGF), epidermal growth factor (EGF),
platelet-derived growth factor (PDGF)-BB, transforming growth factor
(TGF)-ß1, interleukin (IL)-1ß,
12-O-tetradecanoylphorbol-13-acetate (TPA), dexamethasone,
estradiol, and dihydrotestosterone were from Sigma. Rabbit polyclonal
IgG anti-bFGF was from Santa Cruz Biotechnology (Santa Cruz, CA).
Restriction endonucleases and other reagents used for molecular cloning
were from Boehringer Mannheim. Double-stranded DNA probes were
radiolabeled with [32P-
]dCTP (3000 Ci/mmol) from
Amersham International (Little Chalfont, UK) using a commercial
random-priming kit purchased from Amersham International.
Patients and Tumor Specimens
A total of 24 cartilage-forming tumor specimens obtained from
patients diagnosed and treated at Hospital de Jove-Gijón,
Hospital de Cabueñes-Gijón, and Hospital Central de
Asturias-Oviedo (Spain) were analyzed. Sixteen cases corresponded to
ten male and six female patients diagnosed of conventional
chondrosarcoma (Table 1)
. None of the
patients had received chemo- or radiotherapy before the operation. The
age range was 16 to 82 years (mean, 50.7 years). The most frequent site
of involvement was the hand (three cases) followed in frequency by the
femur and humerus (two cases at each site). Chondrosarcomas were graded
according to standard criteria such as cellularity, mitotic activity,
and nuclear atypia.21
In the overall group of sixteen
malignant tumors, thirteen were grade I, two were grade II, and only
one was a mitotically active grade III tumor. The study population also
included eight cases of benign cartilage-forming neoplasms: six
chondromas, one chondroblastoma, and one osteochondroma. The age range
of these patients (six males and two females) was 14 to 68 years (mean,
39.7 years). Benign and malignant tumor specimens were routinely fixed
and decalcified in 0.3 mol/L EDTA (pH 7.5) before embedding in
paraffin. Five-micron sections were cut and stored at room temperature
until use for histological diagnosis or immunohistochemical
analysis.
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Human chondrosarcoma cells HCS-2/8 were derived from a tumor of the proximal part of the humerus of a 72-year-old Japanese man who had received no surgical treatment or chemical or radiation therapy.22,23 The tumor was identified as a well differentiated chondrosarcoma as previously described.22 SW1353 cells were obtained from the American Type Culture Collection (Rockville, MD) and were derived from a primary grade II chondrosarcoma of the right humerus from a 72-year-old Caucasian woman. Cells were routinely maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS), 100 IU/ml penicillin, and 100 µg/ml streptomycin in a humidified atmosphere of 5% CO2. Cells were subcultured every 2 weeks by incubation at 37°C for 2 minutes with 0.0125% trypsin in 0.02% EDTA, followed by addition of complete medium, washing, and resuspension in fresh medium. For most experiments, approximately 5 x 105 cells were plated out in 100-mm dishes and transferred to serum-free DMEM for 24 hours and then exposed to the different growth factors, cytokines, and tumor promoters at the concentrations and for the times indicated.
Northern Blot Analysis
Total RNA was isolated from HCS-2/8 cells by the guanidinium isothiocyanate procedure according to Chomczynski and Sacchi,24 separated by electrophoresis in 1% agarose-formaldehyde gels, and blotted onto Hybond N nylon filters (Amersham International). Filters containing 20 µg of total RNA were prehybridized at 42°C for 3 hours in 50% formamide, 5X SSPE (1X SSPE contains 150 mmol/L NaCl, 10 mmol/L NaH2PO4, 1 mmol/L EDTA, pH 7.4), 10X Denhardt's, 2% SDS, and 100 µg/ml denatured herring sperm DNA and then hybridized with radiolabeled collagenase-3 full-length cDNA for 20 hours under the same conditions. Filters were washed with 0.1X SSC, 0.1% SDS for 2 hours at 50°C and exposed to autoradiography. RNA integrity and equal loading were assessed by hybridization with a ß-actin probe.
Reverse Transcription and PCR Amplification of RNA from Human Chondrosarcoma Cells
Total RNA from HCS-2/8 or SW1353 chondrosarcoma cells, or from a primary chondrosarcoma was used for cDNA synthesis with the RNA polymerase chain reaction (PCR) kit from Perkin-Elmer/Cetus (Norwalk, CT). The primary chondrosarcoma used in this experiment corresponded to a grade II tumor of the scapula of a 35-year-old man. After RNA isolation and reverse transcription (RT) using 1 µg of total RNA and random hexamers as primer, the whole mixture was used for PCR with two oligonucleotides (5'-CCTCCTGGGCCAAATTATGGAG-3' and 5'-CAGCTCCGCATCAACCTGCTG-3') specific for collagenase-3. As a positive control, we performed RT-PCR of the different RNA samples with two primers (5'-GTGGGGCCGCTCTAGGCAC-3' and 5'-TTTGATGTCACGCACGATTT-3') specific for human actin. The PCR amplifications were carried out in a GeneAmp 2400 PCR system from Perkin-Elmer for 40 cycles of denaturation (95°C for 2 minutes), annealing (64°C for 30 seconds), and extension (72°C for 30 seconds). The PCR products were analyzed in 2% agarose gels, cloned in pUC18, and sequenced by the dideoxy terminator using the Sequenase version 2.0 kit (United States Biochemical Corp., Cleveland, OH).
Western Blot Analysis
Conditioned media were obtained after incubation of HCS-2/8 in serum-free DMEM for 48 hours or supplemented with bFGF, filtered, and dialyzed in an Amicon Centricon-10 microconcentrator (Amicon, Beverly, MA). Proteins from conditioned medium were separated by polyacrylamide gel electrophoresis (PAGE) under denaturing and reducing conditions and transferred to nitrocellulose membranes (Amersham International). After blocking in PBS containing 0.1% Tween-20 (PT buffer) and 5% low-fat dried milk, the membranes were incubated with 0.1 µg/ml monoclonal antibody 181-15A12 raised against recombinant human collagenase-3 (kindly provided by K. Iwata, Fuji Chemical Industries, Takaoka, Japan). After extensive washing of the membranes with PT buffer, they were incubated with a horseradish-peroxidase-conjugated goat antiserum against mouse IgGs diluted 1:20,000 in PT containing 3% low-fat dried milk. The membranes were washed and developed with a horseradish peroxidase chemiluminescence detection reagent (ECL system, Amersham International).
Immunohistochemical Analysis
Immunohistochemical assays were performed on 5-µm, formalin-fixed, paraffin-embedded tissue sections using the streptavidin-biotin method. After routine deparaffinization, sections were treated with 0.3% H2O2 in methanol for 30 minutes to block endogenous peroxidase activity and then washed with three changes of 0.01 mol/L PBS for 5 minutes each and treated with 1% normal serum for 30 minutes. Sections were incubated in a moist chamber for 12 hours at 4°C with monoclonal antibody 181-14G11 against collagenase-3 (2.5 µg/ml). They were then washed with several baths of PBS and incubated sequentially with biotinylated goat anti-mouse antibody (Biomeda Corp., Foster City, CA) and peroxidase-conjugated streptavidin (Biomeda Corp.) for 30 minutes at room temperature in a moist chamber, washing with PBS between incubations. After washes, sections were treated with a solution containing 0.66 mol/L 3,3'-diaminobenzidine and 2 mmol/L H2O2 in 50 mmol/L Tris/HCl, pH 7.6. Sections were finally counterstained for nuclei with hematoxylin, dehydrated, and mounted with Eukitt. The purity and identity of the antigen used for developing monoclonal antibodies was assessed by automatic Edman degradation of the recombinant human collagenase-3. Specificity of staining was determined using controls that involved incubation of tissues with buffer alone or with an equal amount of IgG from nonimmunized animals. In both cases, there was no significant staining. Furthermore, immunostaining was completely abolished by antiserum preincubation with purified collagenase-3, produced in a vaccinia virus expression system as previously described.1 Immunohistochemical analysis using rabbit polyclonal anti-bFGF IgGs (0.5 µg/ml; Santa Cruz Biotechnology) was performed in the same way, except that the secondary antibody was a biotinylated goat anti-rabbit antibody (Biomeda Corp). The rabbit anti-bFGF was raised against a synthetic peptide corresponding to positions 40 to 63 in the amino acid sequence of human bFGF. This antiserum did not show cross-reactivity with aFGF (FGF-1). Quantitation of immunoreactivity was performed using an Olympus light microscope interfaced via a Sony camera to an image analysis system (Qwin Pro, Leica, Wetzlar, Germany). The percentages of collagenase-3- and bFGF-immunopositive cells were obtained from 20 random fields per case/section using a 10x objective lens. Semiquantitative estimation of both collagenase-3 and bFGF immunostaining intensities was made in immunopositive cells by arbitrarily assigning +++ when cytoplasmic immunostaining appeared dark brown, ++ when it appeared reddish, and + when it appeared yellowish. Statistical analysis was performed with an SPSS statistical package.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To evaluate the possibility that human collagenase-3 is produced
by chondrosarcoma cells, HCS-2/8 cells derived from a well
differentiated chondrosarcoma were analyzed by Northern blot using as a
probe the complete cDNA encoding this enzyme. As illustrated in Figure 1A
, two transcripts of approximately 3.0
and 2.5 kb were detected, albeit at low levels, in total RNA extracted
from untreated HCS-2/8 cells. The size of these mRNA bands agrees
perfectly with that of the two major collagenase-3 transcripts
identified in breast carcinomas,1
squamous cell carcinomas
of the head and neck,13,14
and articular cartilage from
arthritic patients.6-8
Then, and to study the putative
factors that could stimulate the expression of collagenase-3 in human
chondrosarcoma cells, HCS-2/8 cells were incubated for 24 hours in the
presence of a variety of growth factors, cytokines, steroid hormones,
and tumor promoters, and total RNA was subjected to Northern blot
analysis using the same collagenase-3 cDNA probe as above. As shown in
Figure 1A
, bFGF strongly induced the accumulation of the two
collagenase-3 mRNA transcripts, resulting in a sevenfold increase over
the basal levels observed in the unstimulated cells. The bFGF-mediated
induction of collagenase-3 expression in chondrosarcoma cells was also
observed in SW1353 cells, a commercially available human chondrosarcoma
cell line. However, the level of expression of collagenase-3 in these
cells was lower than in HCS-2/8 cells, and RT-PCR was required to
increase the sensitivity of detection (Figure 1B)
. A band of the
expected size (392 bp), the identity of which was confirmed by
nucleotide sequencing, was amplified from RNA obtained of HCS-2/8 and
SW1353 cells treated with bFGF as well as from RNA of a primary
chondrosarcoma. Nevertheless, the amount of collagenase-3 RNA amplified
from SW1353 cells was consistently lower than that detected in HCS-2/8
cells. These variations could be due to the fact that SW1353 have
partially lost their differentiated chondrocyte phenotype, displaying
some characteristics typical of fibroblastic cells. Consequently, all
subsequent studies described in the present work were performed with
HCS-2/8 cells.
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revealed
that the tumor promoter TPA, a potent inducer of most MMPs in a wide
variety of cell types, also induced collagenase-3 expression in HCS-2/8
cells. However, IL-1ß did not exhibit any stimulating effect on
collagenase-3 expression by HCS-2/8 cells, despite that this
pro-inflammatory cytokine has been found to induce collagenase-3
production by normal articular chondrocytes as well as by chondrocytes
from osteoarthritic patients.5-7
The reason for this
observation is unclear as IL-1 receptor (type I) is normally expressed
in these cells. Nevertheless, the finding that HCS-2/8 cells produce
truncated forms of IL-1 could suggest that endogenous abnormal IL-1 may
occupy the receptor, thus blocking the effect of exogenously added
cytokines (M. Takigawa, unpublished results). Figure 1A
also shows that
dexamethasone displayed a down-regulatory effect on collagenase-3
expression in HCS-2/8 cells, whereas the remaining analyzed factors
including EGF, PDGF-BB, TGB-ß, estradiol, and dihydrotestosterone did
not show any effect on the expression of this metalloproteinase by
chondrosarcoma cells. bFGF Induces Collagenase-3 Expression in Chondrosarcoma Cells through a Mechanism Independent of the de Novo Protein Synthesis
As the above results identified bFGF as a potent inducer of
collagenase-3 expression in human chondrosarcoma cells, we undertook a
preliminary analysis of the molecular mechanisms and signal
transduction pathways mediating this effect. To this purpose, we first
examined by Northern blot the time course of the bFGF-induced
up-regulation of collagenase-3 expression in HCS-2/8 cells. As can be
seen in Figure 2
, there was a consistent
increase with time in the steady-state collagenase-3 mRNA levels from
cells treated with 10 ng/ml bFGF. The up-regulatory effect was already
detectable after 6 hours, being maximal 24 hours after bFGF treatment
(sixfold over the control cells), and remaining constant for at least
72 hours. The possibility that the induction of collagenase-3 mRNA
levels by bFGF was dose dependent was also examined by Northern blot
hybridization, and the results obtained are shown in Figure 3
. As can be seen, incubation of the
cells with 10 ng/ml bFGF resulted in a maximal induction of
approximately sevenfold over the control cells, whereas concentrations
as low as 0.1 ng/ml still produced an accumulation of collagenase-3
mRNA of approximately twofold over the control. To determine whether
the up-regulating effect of bFGF on collagenase-3 mRNA levels was also
reflected at the protein level in HCS-2/8 cells, we performed Western
blot analysis with conditioned medium from cells treated with 10 ng/ml
bFGF for 48 hours. As shown in Figure 4, a
clear immunoreactive band of the expected molecular mass
(approximately 60 kd) was detected in the 25-fold concentrated
conditioned medium of HCS-2/8 cells, but not in the medium from
untreated cells.
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, incubation of these cells
with 10 µg/ml cycloheximide (added 1 hour before bFGF) did not
prevent the bFGF-induced accumulation of collagenase-3 mRNA, despite
the fact that this treatment caused an extensive inhibition in overall
protein synthesis. We therefore conclude that the induction of
collagenase-3 expression by bFGF was independent of the synthesis of
proteins de novo. By contrast, it is remarkable that a
superinduction effect on collagenase-3 mRNA was observed as a
consequence of the cycloheximide treatment, suggesting the occurrence
in HCS-2/8 cells of a protein factor that acts as a repressor of
collagenase-3 gene transcription or an increase in the stability of
mRNA transcripts. A similar explanation has been pointed out for the
rat collagenase-3 superinduction observed after addition of
cycloheximide to osteoblastic cells treated with PDGF-BB or retinoic
acid.25,26
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In an attempt to extend the above in vitro observations
derived from studies with a chondrosarcoma cell line, we decided to
investigate the in vivo presence of collagenase-3 in a
series of the most frequent benign and malignant cartilage-forming
neoplasms. A total of 8 benign lesions and 16 malignant chondrosarcomas
were analyzed by immunohistochemical staining with a monoclonal
antibody (181-14G11) raised against recombinant human collagenase-3.
The monoclonal antibody specificity had been previously confirmed by
Western blot analysis of collagenase-3 and a series of different MMPs,
including collagenase-1, collagenase-2, stromelysin-1, stromelysin-2,
gelatinase-A, gelatinase-B, matrilysin, and MT1-MMP. A single band was
detected with collagenase-3, but no immunoreactive band with the
181-14G1 antibody was detected with any of the remaining MMPs (H.
Tamei, I. Azumano, K. Iwata, Y. Yoshihara, C. López-Otín,
F. Vizoso, V. Knäuper, and G. Murphy, submitted for publication).
Immunohistochemical analysis of malignant chondrosarcomas with this
monoclonal antibody revealed that all of them were positive for
collagenase-3 expression (Figure 6, A and B
, and Table 1
). Specificity of immunostaining in all cases was
determined by using controls that involved incubation of tissue
sections with buffer alone or with an equal amount of IgG from
nonimmunized animals. In addition, immunostaining was completely
abolished by antiserum preincubation with purified recombinant
collagenase-3 (Figure 6C
and data not shown). The immunoreactive
pattern of collagenase-3 on chondrosarcoma sections was somewhat
variable in both the intensity and percentage of positive cells (Table 1)
. However, it was common to find strong immunoreactivity in the
small-sized and individually arranged chondrocytes located near blood
vessels (Figure 6, A and B)
. Immunoreactivity for collagenase-3 was
also present, but at lesser extent and intensity, in chondrocytes of
larger size arranged in groups or cell nests (Figure 6, A and B)
. By
contrast, immunohistochemical analysis of benign cartilage-forming
tumors using the same monoclonal antibody as above revealed that most
of them were negative for collagenase-3 expression (Figure 6D)
. In
fact, only two of eight analyzed lesions (one chondroma and one
chondroblastoma) showed some collagenase-3 immunoreactivity.
Representative examples of positively stained malignant chondrosarcomas
and negative benign tumors and controls are shown in Figure 6, A–D
.
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, and in the
representative example depicted in Figure 7, C and D
, a clear bFGF immunoreactivity
was detected in all analyzed chondrosarcomas as well as in the
chondroblastoma case positive for collagenase-3. bFGF immunoreactivity
could be also detected in normal non-tumor-involved cartilage, but its
expression was very low when compared with that observed in
chondrosarcomas (data not shown). Statistical analysis confirmed a
significant positive correlation between collagenase-3 and bFGF
expression values in chondrosarcomas (r = 0.81;
P < 0.0001). It is also worthwhile mentioning that the
pattern of bFGF localization in these chondrosarcoma sections was
similar to that of collagenase-3 (Figure 7, A and B)
, as both proteins
were usually found in small-sized chondrocytes located near blood
vessels. However, immunoreactive cells for bFGF were not coincidental
with those positive for collagenase-3, suggesting that the putative
up-regulatory effect of bFGF on collagenase-3 might be mediated through
a paracrine mechanism.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The finding that collagenase-3 is produced by human chondrosarcomas extends the pattern of tumor types with ability to produce this MMP. In fact, the enzyme was originally cloned from breast carcinomas on the basis of its differential expression in tumors versus normal mammary tissues.1 Subsequently, Heppner et al27 confirmed that collagenase-3 was produced by approximately 30% of human breast carcinomas. More recently, it has been reported that collagenase-3 is overexpressed in a significant number of squamous cell carcinomas of the head and neck, and this expression is associated with increased local invasion of the tumors.13,14 The results obtained in the present study demonstrating the production of this enzyme by human chondrosarcomas suggest that collagenase-3 may be associated with a higher number of malignancies than originally thought. In addition, the observation that this enzyme is produced by all chondrosarcomas analyzed in the present work, but not by a significant number of benign lesions or by normal cartilage in adult tissues,4-9 suggests that this up-regulation may be somewhat linked to the malignant transformation and contribute to the progression of these tumors. This possibility is consistent with previous clinical data showing the production of collagenase-3 by aggressive breast and laryngeal carcinomas as well as with the biochemical properties of this enzyme, which has been characterized as a potent proteinase with a wide substrate specificity, including a preferential degrading activity on type II collagen.2,6,10 As this collagen type is the most abundant in articular cartilage, the widespread occurrence of a degrading enzyme as collagenase-3 in malignant tumors involving these cells may be a major determinant for facilitating cartilage destruction, thus allowing tumor progression in a similar fashion to that proposed for other MMPs in different tumor types.27-30 In this regard, it is of interest that, despite the fact that all analyzed chondrosarcomas were positive for collagenase-3 expression, there were clear variations in the percentage of positive cells and the intensity of staining. These variations could be associated with the occurrence of tumors with different clinical outcome. Preliminary analysis aimed at looking for a putative relationship between collagenase-3 levels and a series of biological and clinico-pathological characteristics did not reveal any apparent association, although the limited number of samples analyzed in this study precludes the ability to obtain definitive conclusions. Additional studies with a large number of samples will be required to clarify whether expression levels of collagenase-3 in human chondrosarcomas may help to identify tumors differing in their biological and/or clinical characteristics. Similarly, it will be of interest to examine whether the small percentage of benign lesions producing collagenase-3 could be indicative of an increased risk of malignant transformation in these patients. In fact, it has been previously reported that malignant change can occur in a benign cartilage-forming tumor and that occasional chondroblastomas lacking any unusual histological features have developed distant metastases and have even proved fatal.19,20
In this work, and as a previous step to identify potential factors that
could be responsible for the observed up-regulation of collagenase-3
expression in human chondrosarcomas, we have also examined the ability
of different cytokines and growth factors to induce this MMP in
chondrosarcoma cells. This analysis revealed that bFGF, a member of the
heparin-binding growth factor family with multiple biological
functions,31
was a strong collagenase-3 inducer in these
cells. By contrast, a series of factors, such as IL-1ß, TGF-ß, or
TNF-, which have been previously found to play important roles in
the induction of collagenase-3 in fibroblasts, keratinocytes, or
primary chondrocytes,32-35
did not show any significant
up-regulatory effect on collagenase-3 production by chondrosarcoma
cells. These data appear to indicate that the positive effect of bFGF
on collagenase-3 expression in these cells is rather specific. In fact,
bFGF is unable to induce collagenase-3 expression in a number of
analyzed cell lines, including fibroblasts, epithelial breast cancer
cells, or keratinocytes,14,34,35
although this growth
factor does induce rat interstitial collagenase, the murine homologue
of human collagenase-3, in bone cells.36,37
It is also
worthwhile mentioning that, despite the fact that bFGF has been
previously shown to induce or to inhibit the expression of different
human MMPs in various cell types, including fibroblasts,38
endothelial cells,39
smooth muscle cells,40
monocytes,41
and keratinocytes,42
our findings
represent the first report of induction of a human MMP family member by
this growth factor in chondrosarcoma cells. Additional analysis of the
bFGF-induced expression of human collagenase-3 in these cells revealed
that its effect was time and dose dependent, but independent of the
de novo synthesis of proteins. Nevertheless, it was unclear
whether these in vitro effects of this growth factor on
collagenase-3 expression could reflect its activity in vivo,
thus making bFGF a potential candidate for inducing expression of this
MMP in human chondrosarcomas. In this regard, it is of interest that
the presence of bFGF has been described in a variety of human cancers,
including breast, endometrial, esophageal, ovarian, renal, uterine
cervical, colorectal, pancreatic, or prostatic
adenocarcinomas.43-51
In addition, bFGF levels are usually
higher in highly metastatic tumors than in those less
aggressive.48-51
However, although bFGF was originally
purified from rat chondrosarcoma cells,52
no data were
available on the occurrence of this growth factor in human
chondrosarcomas. The finding that all chondrosarcomas analyzed in the
present work are bFGF immunoreactive provides additional support to the
proposal that this growth factor may be a modulator of collagenase-3
expression in these tumors. Interestingly, the patterns of intratumor
localization of collagenase-3- and bFGF-positive cells are not
superimposable. Thus, collagenase-3 was detected in the vicinity of
bFGF-producing chondrocytes, but very few cells showed concomitant
production of both proteins. These observations suggest that the
stimulatory effect of bFGF on the expression of collagenase-3 could be
exerted through a paracrine signaling pathway rather than through an
autocrine mechanism within the same cell. Finally, it should be
mentioned that the precise molecular mechanisms responsible for
collagenase-3 up-regulation in chondrosarcoma cells in response to bFGF
have not yet been elucidated, although it is likely that members of the
FGF receptor family may be involved.31,53
The availability
of a phenotypically stable chondrosarcoma cell line with ability to
produce collagenase-3 after bFGF treatment will be very helpful to
further evaluate the precise mechanisms mediating the bFGF-elicited
induction of this MMP gene in human chondrosarcomas. These studies may
also contribute to a better understanding of the pathogenesis of these
tumors that constitute a significant percentage of all malignant tumors
arising from the skeletal system, but the biological properties of
which remain largely uncharacterized.
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Acknowledgements |
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Footnotes |
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Supported by grants from Comisión Interministerial de Ciencia y Tecnología (SAF94–0892), EU-BIOMED II (BMH4-CT96–0017), and Glaxo-Wellcome, Spain. M. Balbín is recipient of the Severo Ochoa fellowship from Ayuntamiento de Oviedo-Asturias.
Accepted for publication April 2, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and transforming growth factor ß. Cell Growth Differ 1997, 8:243-250[Abstract]
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Y. Sun, J. M. Cheung, J. Martel-Pelletier, J. P. Pelletier, L. Wenger, R. D. Altman, D. S. Howell, and H. S. Cheung Wild Type and Mutant p53 Differentially Regulate the Gene Expression of Human Collagenase-3 (hMMP-13) J. Biol. Chem., April 6, 2000; 275(15): 11327 - 11332. [Abstract] [Full Text] [PDF]
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 R. K. Nuttall and T. G. Kennedy Epidermal Growth Factor and Basic Fibroblast Growth Factor Increase the Production of Matrix Metalloproteinases during in Vitro Decidualization of Rat Endometrial Stromal Cells Endocrinology, February 1, 2000; 141(2): 629 - 636. [Abstract] [Full Text] [PDF]
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R. M. Korah, V. Sysounthone, Y. Golowa, and R. Wieder Basic Fibroblast Growth Factor Confers a Less Malignant Phenotype in MDA-MB-231 Human Breast Cancer Cells Cancer Res., February 1, 2000; 60(3): 733 - 740. [Abstract] [Full Text]
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 N Johansson, R Ala-aho, V Uitto, R Grenman, N. Fusenig, C Lopez-Otin, and V. Kahari Expression of collagenase-3 (MMP-13) and collagenase-1 (MMP-1) by transformed keratinocytes is dependent on the activity of p38 mitogen-activated protein kinase J. Cell Sci., January 1, 2000; 113(2): 227 - 235. [Abstract] [PDF]
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L. Ravanti, L. Hakkinen, H. Larjava, U. Saarialho-Kere, M. Foschi, J. Han, and V.-M. Kahari Transforming Growth Factor-beta Induces Collagenase-3 Expression by Human Gingival Fibroblasts via p38 Mitogen-activated Protein Kinase J. Biol. Chem., December 24, 1999; 274(52): 37292 - 37300. [Abstract] [Full Text] [PDF]
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 M. J. G. Jimenez, M. Balbin, J. M. Lopez, J. Alvarez, T. Komori, and C. Lopez-Otin Collagenase 3 Is a Target of Cbfa1, a Transcription Factor of the runt Gene Family Involved in Bone Formation Mol. Cell. Biol., June 1, 1999; 19(6): 4431 - 4442. [Abstract] [Full Text] [PDF]
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 J. WESTERMARCK and V.-M. KÄHÄRI Regulation of matrix metalloproteinase expression in tumor invasion FASEB J, May 1, 1999; 13(8): 781 - 792. [Abstract] [Full Text]
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 N. Johansson, M. Vaalamo, S. Grenman, S. Hietanen, P. Klemi, U. Saarialho-Kere, and V.-M. Kahari Collagenase-3 (MMP-13) is Expressed by Tumor Cells in Invasive Vulvar Squamous Cell Carcinomas Am. J. Pathol., February 1, 1999; 154(2): 469 - 480. [Abstract] [Full Text] [PDF]
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L. Ravanti, J. Heino, C. Lopez-Otin, and V.-M. Kahari Induction of Collagenase-3 (MMP-13) Expression in Human Skin Fibroblasts by Three-dimensional Collagen Is Mediated by p38 Mitogen-activated Protein Kinase J. Biol. Chem., January 22, 1999; 274(4): 2446 - 2455. [Abstract] [Full Text] [PDF]
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S. K. Winchester, N. Selvamurugan, R. C. D'Alonzo, and N. C. Partridge Developmental Regulation of Collagenase-3 mRNA in Normal, Differentiating Osteoblasts through the Activator Protein-1 and the runt Domain Binding Sites J. Biol. Chem., July 21, 2000; 275(30): 23310 - 23318. [Abstract] [Full Text] [PDF]
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