Celecoxib treatment of fibrous dysplasia (FD) in a human FD cell line and FD-like lesions in mice with protein kinase A (PKA) defects.

Osteochondromyxomas (OMX) in the context of Carney complex (CNC) and fibrous dysplasia (FD)-like lesions (FDLL) in mice, as well as isolated myxomas in humans may be caused by inactivation of PRKAR1A, the gene coding for the type 1a regulatory subunit (R1α) of cAMP-dependent protein kinase (PKA). OMXs and FDLL in mice lacking Prkar1a grow from abnormal proliferation of adult bone stromal cells (aBSCs). Prkar1a and Prkaca (coding for Cα) haploinsufficiency leads to COX2 activation and prostaglandin E2 (PGE2) production that, in turn, activates proliferation of aBSCs. Celecoxib is a cyclooxygenase-2 (COX2) inhibitor. We hypothesized that COX-2 inhibition may have an effect in FD and FDLL. In vitro treatment of a human cell line prepared from a FD patient with Celecoxib resulted in decreased PGE2 and cell proliferation. Treatment of mice haploinsufficient for R1α and Cα with 1500 mg/kg Celecoxib led to decreased PGE2 and proliferation and increased apoptosis, with a corresponding gene expression profile, resulting in dramatic reduction of tumor growth. Furthermore, the treatment improved the organization of cortical bone that was adjacent to the tumor. We conclude that, in vitro and in vivo, Celecoxib had an inhibitory effect on FD cell proliferation and in mouse FDLL structure, respectively. We speculate that COX-2 inhibitors offer an attractive alternative to current treatments for benign tumors such as OMX and FD that, apart from tumor suppression, may mechanically stabilize affected bones.

Constitutive expression of Gsα(R201C) in mice produces a heritable, direct replica of human fibrous dysplasia bone pathology and demonstrates its natural history.

Fibrous dysplasia of bone (FD) is a crippling skeletal disease associated with postzygotic mutations (R201C, R201H) of the gene encoding the α subunit of the stimulatory G protein, Gs. By causing a characteristic structural subversion of bone and bone marrow, the disease results in deformity, hypomineralization, and fracture of the affected bones, with severe morbidity arising in childhood or adolescence. Lack of inheritance of the disease in humans is thought to reflect embryonic lethality of germline-transmitted activating Gsα mutations, which would only survive through somatic mosaicism. We have generated multiple lines of mice that express Gsα(R201C) constitutively and develop an inherited, histopathologically exact replica of human FD. Robust transgene expression in neonatal and embryonic tissues and embryonic stem (ES) cells were associated with normal development of skeletal tissues and differentiation of skeletal cells. As in humans, FD lesions in mice developed only in the postnatal life; a defined spatial and temporal pattern characterized the onset and progression of lesions across the skeleton. In individual bones, lesions developed through a sequence of three distinct histopathological stages: a primary modeling phase defined by endosteal/medullary excess bone formation and normal resorption; a secondary phase, with excess, inappropriate remodeling; and a tertiary fibrous dysplastic phase, which reproduced a full-blown replica of the human bone pathology in mice of age ≥1 year. Gsα mutations are sufficient to cause FD, and are per se compatible with germline transmission and normal embryonic development in mice. Our novel murine lines constitute the first model of FD.

Immunofluorescence localization of prolyl 4-hydroxylase isoenzymes and type I and II collagens in bone tumours: type I enzyme predominates in osteosarcomas and chondrosarcomas, whereas type II enzyme predominates in their benign counterparts.

Prolyl 4-hydroxylase is the key enzyme of synthesis of collagens. Hydroxylation of a sufficient number of proline residues to hydroxyproline is necessary for the stability of triple helices in collagenous proteins, because non-hydroxylated non-triple-helical collagen polypeptide chains are degraded intracellularly. We studied 15 primary chondrosarcomas and osteosarcomas, 17 benign bone tumours and one case of fibrous dysplasia and chordoma using immunofluorescence staining with antibodies against the alpha(I) and alpha(II) subunits of type I and II prolyl 4-hydroxylases, and with antibodies against collagen types I and II. Type I prolyl 4-hydroxylase was found to be the predominant isoenzyme in both types of bone sarcoma, whereas the type II enzyme was more readily expressed by benign tumours. A feature of collagen staining, that was common to both sarcoma types, was that collagen types I and II were mainly found within cancer cells and were rarely present extracellularly. Extracellular collagen staining was more obvious in benign tumours. The results show that expression of prolyl 4-hydroxylase isoenzymes is altered in bone sarcomas as compared with normal bone tissue. Chondrous cells, which normally express mainly the type II isoenzyme, switch their expression pattern to that of type I. The findings provide evidence that type I is the major isoenzyme in malignant bone tumours, and probably in malignant neoplasms in general. The pattern of enzyme expression is considered to be associated with dedifferentiation of cancer cells.

Telomerase activity in benign bone tumors and tumor-like lesions.

To assess the role and status of telomerase activity in benign bone tumors and tumor-like lesions, we performed telomerase assays in four giant cell tumors of bone, four fibrous dysplasias, three osteochondromas, three aneurysmal bone cysts, two osteoblastomas, one juvenile bone cyst and one myositis ossificans. A very sensitive non-radioactive TRAP assay was applied. Low level activity was detected in 7 of 18 tumor samples (38.9%), and high level activity was not detected in any of the cases. Telomerase activity was observed in all patients with osteochondromas, in two of the three aneurysmal bone cysts, in one of the four giant cell tumors of bone and in one of the four fibrous dysplasias, but not in osteoblastomas, juvenile bone cyst and myositis ossificans. Although the origin of this enzyme is still unclear, it might play a role in precancerous immortalization of benign bone tumors. Other possible reasons explaining the occurrence of telomerase activity, such as migrating lymphocytes or contamination of immortalized non-tumor cells, should not be ruled out. Telomerase activity, however, does exist in those samples having no malignant phenotype, for which reason telomerase assays are not always useful for the clinical and diagnostic approach in benign bone tumors. Determination of the telomerase status in benign lesions may contribute to a better understanding of the regulation mechanism of telomerase activity during progression of bone tumors.

Size dependent enzyme activities of multinucleated (osteoclastic) giant cells in bone tumors.

In osteoclastic giant cells of six different tumors of bones and joints (fibrous dysplasia, proliferating giant cell tumor, malignant giant cell tumor, osteosarcoma after chemotherapy, malignant synovioma and Ewing's sarcoma) activities of tartrate-resistant acid phosphatase, NADH-tetrazolium-oxidoreductase and, in three of them, of non-specific esterase are determined by enzyme histochemical methods. Quantitative microphotometry makes it possible to determine relative enzyme activities in the cut sections of giant cells of different sizes. Giant cells of the various tumors reveal similar trends: With an increase in cell size, mean extinctions of NADH-tetrazolium-oxidoreductase and non-specific esterase decrease. Mean extinctions of tartrate-resistant acid phosphatase increase in cells of medium size, whereas the large cells reveal in part low activities. An additional ultrastructural examination of the giant cells in the proliferating giant cell tumor as well as in the osteosarcoma shows morphological signs of degeneration in the large cells. Electron probe microanalysis of the proliferating giant cell tumor exhibits evidence of phagocytosis of Ca and/or Fe containing particles. The similar size dependent reaction pattern of enzymes in osteoclastic giant cells of different tumors favors the concept of a common histogenesis, i.e. a host reaction.

[Fibrous dysplasia and ossifying fibroma. Morphologic criteria]. / Dysplasie fibreuse et fibrome ossifiant. Critères morphologiques.

Among a series of 42 ossifying fibromas and 32 fibrous dysplasias, 5 cases were studied by histoenzymological and electron microscopic methods. The histological study in 2/3 of cases is sufficient for the diagnosis between the two diseases: trabecular bone in a connective tissue with regular collagen fibres in the ossifying fibroma; nodular indented bony areas with irregular collagen frame in fibrous dysplasia. By histoenzymology, the diagnosis between the two affections is not easy: same activity of ATPases and alkaline phosphatases in fibroblasts and osteoblasts. By electron microscopy, the morphology of the two lesions is different: in ossifying fibroma, numerous well-differentiated osteoblasts and large areas of ossification are seen; in fibrous dysplasia, undifferentiated cells are numerous and the collagen frame is irregularly mineralized. This method is also useful for the histogenetic understanding of these two osteopathies.

Histochemical and electron-microscopic aspects of bone tumor diagnosis.

In recent years, histochemistry and electron microscopy have been applied more and more to the investigation of bone tumors. The contributions and limitations of these methods in differential diagnosis are discussed. The levels of glycosaminoglycans in cartilaginous tumors display distinct differences between slow- and fast-growing types. All cartilaginous tumors are poor in phosphatase activity. Demonstration of these enzymes at acid and alkaline pH in bone-forming conditions reveals differences between benign and malignant tumors. Osteosarcomas display a rich activity of both phosphatases in bone-forming and in bone-free regions. Acid phosphatase may play a rôle in the breakdown of the host tissue infiltrated by the tumor. Electron microscopy of bone tumors has brought out some interesting findings. In fibrous dysplasia a particular kind of very fine fibrillar structures was observed besides the regular collagen fibrils. This may indicate retardation of collagen maturation. Cell organelles in benign and malignant bone tumors usually differ quantitatively. They resemble active fibroblasts. In bone- and in cartilage-forming tumors we observed large quantities of microfilaments in the cytoplasm. Nuclear indentations and invaginations probably indicate increased nuclear activity. The intense acid phosphatase activity demonstrated histochemically seems inconsistent with the low number of lysosomes in the cytoplasm of osteosarcoma cells, but other organelles (Golgi apparatus and vesicles) may also contain the enzyme. Virus-like particles have not been observed in human osteosarcomas up to now. Other authors have observed a correlation between the number of cell organelles and the grade of differentiation, but this was not detected in our sample of benign and malignant cartilaginous tumors. Histochemistry and electron microscopy of bone tumors are still in the early stage of material gathering. Some histochemical findings, however, can already be used as diagnostic tools.