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Regular Articles |
From the Laboratorio di Ricerca Oncologica*
andServizio di Anatomia Patologica,
IstitutiOrtopedici Rizzoli, Bologna, Italy; the Dipartimento di PatologiaSperimentale,
Sezione di Ricerca sul Cancro,Università di Bologna, Bologna, Italy; the Laboratoired’Oncologie Virale et Moléculaire,
UFR de Biochimie, Université Paris, Paris, France; and theDepartment of Paediatric Laboratory Medicine,¶
Hospital for Sick Children, Toronto, Canada
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Abstract |
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 Top Abstract Materials and MethodsResultsDiscussionReferences |
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The two best-characterized members of the CCN family of proteins, CYR61
and CTGF, are secreted, extracellular matrix-associated proteins that
regulate different cellular processes, such as adhesion, migration,
mitogenesis, differentiation, and survival. Moreover, they seem to play
a key role in angiogenesis, chondrogenesis, and in the development of
embryonic skeleton, and have been implicated in wound healing,
tumorigenesis, and fibrotic and vascular diseases.2,4
The
mechanisms through which CYR61 and CTGF might act to execute such a
diversity of biological functions are still poorly understood. No
specific cell surface receptors for these two proteins have been
clearly identified to date, although a receptor-ligand complex of
280-kd molecular size has been reported in a human chondrocytic cell
line.5
Recent studies have presented evidence that CYR61
and CTGF are ligands of the integrins
vß3 and
IIbß3,6,7
suggesting that CYR61 and CTGF may function as adhesive signaling
molecules acting through integrin-mediated signaling
pathways.8,9
Compared with Cyr61 and CTGF, CCN3 has been studied to a lesser degree
and its biological role remains primarily unknown. ccn3 gene was
identified as an aberrantly expressed gene in avian nephroblastoma
induced by myeloblastosis-associated virus (MAV).10
ccn3
maps on chromosome 8q24.1 and encodes a protein of 48 kd with a
secreted signal peptide that is associated with the extracellular
matrix.4
It shares a homology of 
50% with CYR61 and
CTGF. Transcription of the ccn3 promoter is down-regulated by the
expression of the Wilms’ tumor suppressor gene WT111
and
the levels of WT-1 and ccn3 RNAs are inversely correlated in human
nephroblastomas,12
suggesting a role for this protein in
kidney development or function.13
However, CCN3 was found
to be expressed in several other tissues, including nervous system,
lung, heart, liver, spleen, thymus, endothelium of blood vessels, and
skeletal muscle.10,13-16
Moreover, overexpression of CCN3
in cartilage and in mesenchymal tumors is starting to become
recognized.17
In Wilms’ tumors, CCN3 is mainly
overexpressed in tumors of predominantly stromal origin12
and is positively correlated with heterotypic muscle
differentiation,13
further suggesting a role of this
protein in the musculoskeletal system. With regard to CCN3 protein
functions, no specific biological activity has yet been reported. In
chicken embryo fibroblasts, the ccn3 gene was expressed only in
quiescent cells and their entry into the cell cycle, as a result of
either the expression of an oncogene (ie, v-src) or serum stimulation
which led to down-regulation of its transcription.18
Based
on this expression pattern and its growth inhibitory
properties,10
ccn3 has been proposed to function as a
negative regulator of the cell growth. Indeed, in humans, the
expression of CCN3 was associated with differentiation of glomerular
podocytes during normal nephrogenesis and with tumor-derived
mesenchymal differentiation (striated muscle and cartilage) in Wilms’
tumors.13
However, a recent article indicated that CCN3
could act as a growth factor, stimulating proliferation of 3T3
fibroblast cells and inducing protein tyrosine
phosphorylation.14
Therefore, the definition of the role
and function(s) of CCN3 in normal and tumoral tissues is still
controversial and intriguing.
In this article, we have analyzed the expression of CCN3 in normal mesenchymal cells as well as in several musculoskeletal tumors. Our findings indicate that CCN3 is expressed in cells of the musculoskeletal system and that this protein likely participates in different cellular processes depending on cell type. It seems, in fact, to be associated with differentiation in rhabdomyosarcoma and cartilage tumors and with increased aggressiveness of Ewing’s sarcoma.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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A panel of 13 osteosarcoma, 12 Ewing’s sarcoma, and 5 rhabdomyosarcoma cell lines were analyzed. The osteosarcoma cell lines Saos-2 and U-2 OS, the Ewing’s sarcoma cell lines SK-ES-1 and RD-ES, the Askin’s tumor cell line SK-N-MC and the alveolar rhabdomyosarcoma cell lines SJ-Rh 30 and SJ-Rh 4 were obtained from the American Type Collection (Rockville, MD). The Ewing’s sarcoma cell lines TC-71 and 6647 were kindly provided by T. J. Triche (Children’s Hospital, Los Angeles, CA). All of the other osteosarcoma cell lines (SARG, MOS, IOR/OS7, IOR/OS9, IOR/OS10, IOR/OS14, IOR/OS15, IOR/OS17, IOR/OS18, IOR/OS19, and IOR/OS20) as well as the Ewing’s sarcoma cell lines here considered (LAP35, IOR/BRZ; IOR/CAR; IOR/NGR; IOR/BER; IOR/RCH; and IOR/CLB) were obtained at the Laboratorio di Ricerca Oncologica, Istituti Ortopedici Rizzoli, Bologna, Italy, and previously characterized.19 The CCA cell line was obtained from a human embryonal rhabdomyosarcoma.20 The RMZ-RC2 cell line was obtained from an alveolar rhabdomyosarcoma.21 The RD/18 cell line is a clone of the commercially available human embryonal rhabdomyosarcoma cell line RD (purchased from Flow Laboratories, VA). The adrenocortical carcinoma NCI-H295R (American Type Collection) were used as positive control for CCN3 detection by Western blotting.17 Cells were routinely cultured in Iscove’s modified Dulbecco’s medium supplemented with 20 U/ml penicillin, 100 µg/ml streptomycin (Sigma, St. Louis, MO), and 10% heat-inactivated fetal calf serum (Biological Industries, Kibbutz Beth Haemek, Israel). Cells were maintained at 37°C in a humidified 5% CO2 atmosphere.
Patients and Tissue Samples
A total of 87 formalin-fixed, paraffin-embedded, musculoskeletal
tumor specimens were selected for the study. They included 8 giant cell
tumors, 2 chondroblastomas, 9 chondrosarcomas, 13 osteosarcomas, 5
rhabdomyosarcomas, and 45 Ewing’s sarcomas. All of the samples were
from primary lesions of previously untreated patients. The histology of
the primary tumors was reviewed by a pathologist with special expertise
in bone tumors (FB). An additional panel of rhabdomyosarcoma cases
(three embryonal and two alveolar) were accessed from the pathology
files of the Hospital for Sick Children. These included both primary
sites and metastases to lung and scalp. The Ewing’s sarcoma patients
were seen at the Istituti Ortopedici Rizzoli between 1983 and 1993 and
treated with three consecutive programs of chemotherapy (REN-1, REN-2,
and REN-3) that have previously been reported in detail.22
Local treatment consisted of surgery only, surgery followed by
radiation therapy, or radiation therapy only, depending on patient age,
the site and the size of the tumor, and the necessity to retain the
greatest level of function of the tumor-affected size. The present
study included 45 patients that were randomly selected for their
different clinical course among the larger series of patients seen at
the Rizzoli Institute during the same period of time, and for whom
tumor samples from the biopsy specimens (obtained before chemotherapy)
were available for immunohistochemical analysis. Fifteen patients were
disease-free at a median follow-up of 145 months (range, 122 to 192
months), 14 patients had developed metachronous lung metastases at a
median time of 19 months after the end of the local treatment, and the
other 16 patients had developed metachronous bone metastases at a
median time of 18 months after the end of the local treatment. Table 1
summarizes the clinical characteristics
of the 45 patients here considered and compares them with the entire
group of patients seen at the Rizzoli Institute in the same period of
time.
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The immunohistochemistry protocol of Chevalier and colleagues13 was used with minor modifications. Five-µm sections from undecalcified, formalin-fixed, paraffin-embedded tissue samples were placed on poly-L-lysine-coated slides (Sigma). The avidin-biotin-peroxidase procedure was used for immunostaining. Briefly, sections were treated sequentially with xylene and ethanol to remove paraffin. Endogenous peroxidase activity was blocked by treatment with 3% hydrogen peroxide in methanol for 30 minutes at room temperature. Antigen retrieval by microwave treatment for 15 minutes in an 800 W microwave oven was performed in citrate solution. A blocking step with normal goat serum (Vector, Burlingame, CA) was used. The primary antibody K19M13 (diluted 1:400) was applied overnight in a moist chamber at 4°C. The following day, the tissue sections were incubated with secondary biotinylated goat anti-rabbit antibody and with avidin-biotin-peroxidase complex (Vector). The final reaction product was revealed by exposure to 0.03% diaminobenzidine (Sigma), and the nuclei were counterstained with Mayer’s hematoxylin. Specimens in which the incubation with the primary antibody had been omitted were used as a negative control. In each experiment, normal kidney tissue was also included as a positive control.
Each case was scored by a pathologist blinded to patient identity. The cases were scored as zero when there was a complete absence of staining for CCN3 protein or when scattered positive cells were observed. The CCN3-positive tumor samples were graded from 1 to 3 according to the distribution of positivity and degree of immunostaining. In particular, score 1 indicated limited positivity (<50% of the specimen) and weak immunostaining; score 2 indicated diffuse positivity (>50% of the specimen) and moderate immunostaining; and score 3 indicated diffuse immunostaining (>75% of the specimen) and strong immunostaining.
Immunofluorescence Analysis of CCN3 Expression
In sarcoma cell lines, CCN3 expression was evaluated by indirect immunofluorescence and cytofluorometric analysis. Briefly, cells from subconfluent cultures were harvested, washed with phosphate-buffered saline (PBS), and fixed with cold methanol for 15 minutes. Cells were incubated with the primary anti-CCN3 polyclonal antibody K19M diluted 1:1200 and, after washing in PBS, with a fluorescein isothiocyanate-conjugated anti-rabbit monoclonal antibody, diluted 1:80 (Technogenetics, Milan, Italy). Cell fluorescence was then evaluated with a FACSCalibur flow cytometer (Becton Dickinson, Mountain View, CA) or, for adherent cells, with a QUIPS-XL image analysis system (Vysis Inc., Downers Grove, IL). Differentiation ability of rhabdomyosarcoma cells was studied on cultures maintained in medium supplemented with 2% horse serum. After 5 days, cells were harvested and analyzed for CCN3 and myosin expression. The percentage of myosin-positive cells was determined after staining with the monoclonal BF-G6 antibody (kindly provided by S. Schiaffino, University of Padova, Padova, Italy), reacting with the embryonic myosin heavy chain.21
Western Blotting
Total protein contained in cell-conditioned medium, harvested 72 hours after the cell seeding, was incubated overnight at 4°C with heparin-Sepharose beads 2.5% because the CCN3 protein is glycosylated and binds to heparin. Samples were electrophoresed on a sodium dodecyl sulfate/12% polyacrylamide gel at 130 V for 60 minutes and transferred at 42 V for 90 minutes to nitrocellulose sheets. Blots were incubated with anti-CCN3 polyclonal antibody K19M antibody (1/500 dilution), and then with the horseradish peroxidase-linked secondary antibody (1/5000 dilution; Amersham, Aylesbury, UK), and revealed by enhanced chemiluminescence Western blotting detection reagents (Amersham).
ccn3 Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR)
Total cellular RNA was isolated from cell cultures and tissue samples. Extraction of RNA was performed by Trizol extraction kit (Life Technologies, Grand Island, NY). cDNA was obtained from 1 µg of RNA by RT-PCR standard methods using Moloney murine leukemia virus reverse transcriptase (Life Technologies) in the presence of dNTPs and oligo-dT. RT-PCR conditions for human ccn3 are: forward primer 5'-CACGGCGGTAGAGGGAGATA-3'; reverse primer 5'-GGGTAAGGCCTCCCAGTGAA-3', annealing temperature 60°C. The reaction was performed for 30 cycles. The PCR product is a fragment of 251 bp, separated by electrophoresis on 2% agarose gel. RT-PCRfor glyceraldehyde-3-phosphate-dehydrogenase gene (Clontech, Palo Alto, CA) was also performed to demonstrate mRNA integrity. In Ewing’ssarcoma cells, EWS fusion transcripts were analyzed by using the primers 22.3, 11.3, and 21.9, previously reported,23,24 annealing temperature 60°C.
Statistical Analysis
Fisher’s exact test was used for frequency data. Correlations were analyzed using Spearman’s test.
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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A high expression of CCN3 was observed in osteoblasts (Figure 1A)
, osteoclasts (Figure 1B)
,
chondrocytes (Figure 1C)
, and muscle skeletal cells (Figure 1D)
.
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A panel of 13 osteosarcoma, 12 Ewing’s sarcoma, and 5
rhabdomyosarcoma cell lines was analyzed. Figure 2
shows the expression of ccn3 mRNA in
these cells by RT-PCR. Jurkat cells were used as a negative
control.14
All of the musculoskeletal cell lines expressed
ccn3 mRNA. However, differences were observed in the level of
expression, with the osteosarcoma cells showing the highest mRNA levels
of expression and the rhabdomyosarcoma cells the lowest. In contrast,
the analysis of ccn3 expression at protein level by indirect
immunofluorescence and cytofluorometric analysis gave somewhat
different results, confirming that ccn3 mRNA and protein expression
patterns showed an overlap but not a direct correlation.13
By cytofluorometric analysis, the highest levels of CCN3 expression
were found in rhabdomyosarcoma cell lines (Figure 3)
. No significant differences were
observed between the alveolar and embryonal subtype. Indirect
immunofluorescent assays on fixed adherent rhabdomyosarcoma cells
indicate the CCN3 protein is widely distributed in the cytoplasm of
mononuclear cells, sometimes associated with perinuclear membrane and
Golgi apparatus, and highly expressed in fusing myoblasts (Figure 1, E and F)
. The association between CCN3 expression and myogenic
differentiation was demonstrated in the RMZ/RC2 cell line, showing a
parallel time-dependent significant increase in the expression of CCN3
and the percentage of myosin-positive cells (data not shown). A
variable expression of CCN3 was observed in osteosarcoma cell lines
(Figure 4)
. Five cell lines were
completely negative; two cell lines were barely positive whereas a weak
but clear positivity was observed in the other six cell lines. The
expression of CCN3 in osteosarcoma cell lines was inversely correlated
with the expression of alkaline phosphatase, a marker of the
osteoblastic differentiation (r = -0.70,
P = 0.01, Spearman’s test; data not shown). Among
Ewing’s sarcoma cell lines, only 3 of 12 showed a weak positivity for
CCN3 (Figure 5)
. Ewing’s sarcoma cells
carried specific chromosomal translocations.25
The
expression of the different chimeric products in the cell lines here
considered is reported on Table 2
. No
significant correlation between expression of CCN3 and the type of
hybrid transcripts was observed. However, it is interesting to note
that all of the three CCN3-positive cell lines showed the expression of
the less frequent types of chimeric products, EWS/FLI-1 type 2 and
EWS/ERG.25
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. In parallel, immunohistochemistry
was also made on formalin-fixed, paraffin-embedded CCN3-positive or
-negative Ewing’s sarcoma cells to be sure that this technique may be
reliably used in the analysis of tissue samples (Figure 6, B and C)
.
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The expression of ccn3 mRNA was investigated by RT-PCR in a
representative panel of frozen musculoskeletal tumor tissue samples,
including 5 giant cell tumors, 1 chondroblastoma, 6 chondrosarcomas, 10
osteosarcomas, 9 Ewing’s sarcomas, and 1 rhabdomyosarcoma (Figure 7)
. The findings obtained were in
agreement with what was previously observed in normal mesenchymal cells
and musculoskeletal tumor cell lines. In particular, giant cell tumor,
a heterogeneous benign neoplasm in which variable numbers of
osteoclast-like multinucleated giant-cells are dispersed among
mononuclear cells of uncertain histogenesis, showed a generally high
expression of ccn3 mRNA. Chondroblastoma, a benign tumor composed of
chondroblasts, and the three cases of chondrosarcoma, a malignant tumor
differentiating in cartilage, expressed high levels of ccn3 mRNA.
Accordingly to the data obtained in cell lines by RT-PCR, the
expression of ccn3 mRNA was homogeneously high in osteosarcoma,
variable in Ewing’s sarcoma, and low in rhabdomyosarcoma.
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. Central
chondrosarcoma stained positively for CCN3. However, differences in the
level of expression were observed among the samples and inside the
single tissue samples. In general, the undifferentiated,
noncartilaginous components showed a weaker immunostaining, whereas the
well-differentiated cartilage component expressed CCN3 at extremely
high levels (Figure 8, C and D)
. The two cases of peripheral
chondrosarcoma appeared well differentiated and show a very high
expression of CCN3.
All of the rhabdomyosarcomas analyzed here overexpressed CCN3 at high
levels. We had previously reported that by in situ
hybridization ccn3 mRNA seemed comparable to protein
levels.26
The expression level of CCN3 seemed to be higher
in the alveolar rhabdomyosarcoma than in the subtype of embryonal
origin (Figure 8, E and F)
. In embryonal rhabdomyosarcoma,
well-differentiated cells showed a high expression of the protein, in
agreement with previous observations indicating a relationship between
CCN3 expression.13,26
With regard to osteosarcoma, all of the primary tumors and lung
metastases of patients with osteosarcoma were clearly positive for the
protein (Figure 8, G and H)
.
In contrast with the other mesenchymal tumors, the great majority of
Ewing’s sarcoma samples were completely negative for CCN3 protein.
However, a moderate or even high expression was observed in a few
percentages of cases (Figure 8, I and J)
.
Prognostic Relevance of CCN3 Expression in Ewing’s Sarcoma
The prognostic significance of CCN3 expression in Ewing’s sarcoma was evaluated by immunohistochemistry on 45 primary tumors that were randomly selected for their different clinical course among a homogeneously treated series of patients referred to the Rizzoli Institute between 1983 and 1993. The patients were followed-up for a minimum of 91 months (median, 144 months). Results were updated in December 2000: 15 patients were free of disease, 16 patients had developed bone metastases, and 14 patients had lung metastases. CCN3 expression was found in 16 of 45 tissue samples (35%). In particular, we did not observe any expression of CCN3 in the 15 primary tumors of patients that did not show clinical progression. In contrast, 16 of the 30 primary tumors that developed lung or bone metastases were positive for CCN3 immunostaining (P < 0.001, Fisher’s test). In particular, five cases were scored as 1, five as 2, and six as 3 (see Materials and Methods). In the group of tumors that gave metastases, the incidence of CCN3 expression was slightly higher in the primary tumors that developed lung metastases (9 of 14, 64%), compared with tumors that developed bone metastases (7 of 16, 44%). In both of the two subgroups, the incidence of CCN3 positivity was significantly different from that observed in the tumors that did not show evidence of clinical progression (9 of 14 versus 0 of 15, P < 0.001; 7 of 16 versus 0 of 15, P = 0.007; Fisher’s test).
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Expression of CCN3 in the Musculoskeletal System and Derived Tumors
Similar to CYR61 and CTGF,27,28 CCN3 was highly expressed in normal human mesenchymal cells of the musculoskeletal system, such as osteoblasts, osteoclasts, chondrocytes, and skeletal muscle cells, further supporting the idea that the CCN genes might be heretofore underestimated regulators of normal mammalian chondrogenesis and musculoskeletal development. In pathological conditions, expression of CCN3 was widely observed in musculoskeletal tumors, with the only notably exception of bone Ewing’s sarcoma. The expression of CCN3 was high in the osteoclast-like, multinucleated giant cells featuring giant cell tumors and infiltrating chondroblastomas.
In the cartilage tumors, the expression of this protein was lower in the undifferentiated, mixoid component of the tumor and extremely high in the cartilaginous, differentiated areas, indicating an association between CCN3 expression and cartilage differentiation. This is in agreement with findings obtained in normal tissues.17 In fact, in situ hybridization and immunohistochemistry performed on developing chicken and human embryos have revealed a strong expression of ccn3 RNA and proteins at sites of chondrogenesis.16,17 Moreover, by using mesenchymal cells that are able to undergo chondrocytic differentiation in vitro, it was possible to establish that CCN3 and CTGF were required at late stages of the chondrogenesis/osteogenesis differentiation process, whereas CYR61 was required at early stages. Whether the differential expression of these three CCN genes is temporally related remains to be established.
Rhabdomyosarcoma expressed high amounts of CCN3, either in cell lines and clinical samples. The expression was particularly evident in the most differentiated cells, supporting the existence of an association between CCN3 expression and tumor differentiation in rhabdomyosarcoma. These findings reflect previous data obtained in normal tissues. Muscle was found to be the predominant mesodermal component expressing CCN3 in human embryos and myotubes and fusing myoblasts were identified as major sites of CCN3 expression in skeletal muscle.17
In osteosarcoma, CCN3 was expressed in all of the tissue samples and in the 61% of the cell lines. Interestingly, in osteosarcoma cell lines, we observed an inverse correlation between expression of CCN3 and alkaline phosphatase, an early marker of osteoblastic differentiation29 that was found to be associated with a loss of aggressiveness of osteosarcoma cells.30,31 Based on these findings and on the consideration that MAV can also induce osteopetrosis, an abnormal proliferation of osteoblasts leading to severe bone diseases, it is tempting to speculate that CCN3 may play a role in sustaining the growth of osteoblast-like cells.
Ewing’s Sarcoma and CCN3: A Potential Prognostic Indicator of Metastatic Potential
In contrast with the other mesenchymal tumors here examined, Ewing’s sarcoma, the second most frequent tumor of bone, showed the expression of CCN3 protein only in the minority of the cases. This could reflect the different origin of Ewing’s sarcoma with respect to the other musculoskeletal tumors. The normal counterpart of Ewing’s sarcoma is still unknown and this tumor has been the metaphorical "Holy Grail" of pathology in the quest to establish its histogenesis. Although different hypothesis have been raised during the last decades,32 passing through the suggestion of an hematopoietic origin to the idea of a neural origin, the current prevalent opinion is that Ewing’s sarcoma derives from a pluripotential uncommitted mesenchymal cell that can variably present neural differentiation, epithelial features, as well as mesenchymal characteristics. The very undifferentiated nature of the Ewing’s sarcoma precursor may be responsible for the lower rate of overexpression of CCN3. Whether or not the subgroup of Ewing’s sarcoma samples overexpressing CCN3 identifies a specific pathological variety of this neoplasm will be the subject of forthcoming studies. In this article, we report on a prognostic significance of CCN3 expression in Ewing’s sarcoma. The expression of this protein is, in fact, significantly associated with a higher risk of developing lung and/or bone metastases. Although these findings need to be confirmed in a larger series of patients, they are extremely interesting and promising because CCN3 may be the first biological marker of prognosis for Ewing’s sarcoma patients.25 Based on the observation that in Ewing’s sarcoma cell lines the expression of CCN3 was found only in those cell lines carrying a type 2 EWS/FLI-1 or a EWS/ERG fusion transcripts, molecular markers associated with worse outcome,33,34 it is tempting to speculate about a possible association between these two parameters. A future parallel analysis of CCN3 and the type of fusion transcripts in Ewing’s sarcoma clinical samples will settle this question.
In conclusion, CCN3 protein is generally expressed in the cells of the musculoskeletal system, indicating that this protein may play a role both in normal and pathological conditions. However, the regulation of CCN3 expression varies in the different neoplasms and depends on the type of cells. An association between CCN3 expression and tumor differentiation was observed in rhabdomyosarcoma and cartilage tumors, whereas in pronounced contrast and apparent contradiction with these observations, the expression of CCN3 correlated with the metastatic potential of tumor cells in the case of Ewing’s sarcoma. Therefore, it seems that the expression of CCN3 is altered in many tumors and that high amounts of the protein could be associated with either differentiation (Wilms’ tumor, glioblastoma, neuroblastoma, rhabdomyosarcoma, and chondrosarcoma) or with increased proliferation and metastases (Ewing’s sarcoma, prostate cancer, and renal cell carcinoma).17 This complex and conflicting situation is common to other members of the CCN genes and likely reflects the complexity of functions of these proteins. We believe that additional investigations are required to fully appraise the biological role of CCN3 in tumor progression and malignancy.
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Footnotes |
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Supported by grants from the Italian Association for Cancer Research, the Italian Ministry for University and Research, the Rizzoli Institute, the Italian Ministry of Health (Ricerca Finalizzata), the Association pour la Recherche sur le Cancer and Ligue Nationale contre le Cancer (Comités du Cher et de l’Indre) (to B. P.); a fellowship from UICC International Cancer Technology Transfer (to S.B.); a fellowship from the Italian Fondation for Cancer Research (to V. C.); a fellowship from the Rizzoli Institute (to R. S.); and a Ph.D. fellowship from the Italian Ministry for University and Research (to A. A.).
Accepted for publication November 14, 2001.
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D. M. French, R. J. Kaul, A. L. D'Souza, C. W. Crowley, M. Bao, G. D. Frantz, E. H. Filvaroff, and L. Desnoyers WISP-1 Is an Osteoblastic Regulator Expressed During Skeletal Development and Fracture Repair Am. J. Pathol., September 1, 2004; 165(3): 855 - 867. [Abstract] [Full Text] [PDF]
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