Modern Interpretation of Giant Cell Tumor of Bone: Predominantly Osteoclastogenic Stromal Tumor

Owing to striking features of numerous multinucleated cells and bone destruction, giant cell tumor (GCT) of bone, often called as osteoclastoma, has drawn major attractions from orthopaedic surgeons, pathologists, and radiologists. The name GCT or osteoclastoma gives a false impression of a tumor comprising of proliferating osteoclasts or osteoclast precursors. The underlying mechanisms for excessive osteoclastogenesis are intriguing and GCT has served as an exciting disease model representing a paradigm of osteoclastogenesis for bone biologists. The modern interpretation of GCT is predominantly osteoclastogenic stromal cell tumors of mesenchymal origin. A diverse array of inflammatory cytokines and chemokines disrupts osteoblastic differentiation and promotes the formation of excessive multi-nucleated osteoclastic cells. Pro-osteoclastogenic cytokines such as receptor activator of nuclear factor kappa-B ligand (RANKL), interleukin (IL)-6, and tumor necrosis factor (TNF) as well as monocyte-recruiting chemokines such as stromal cell-derived factor-1 (SDF-1) and monocyte chemoattractant protein (MCP)-1 participate in unfavorable osteoclastogenesis and bone destruction. This model represents a self-sufficient osteoclastogenic paracrine loop in a localized area. Consistent with this paradigm, a recombinant RANK-Fc protein and bisphosphonates are currently being tried for GCT treatment in addition to surgical excision and conventional topical adjuvant therapies.     

PTHrP increases RANKL expression by stromal cells from giant cell tumor of bone

Giant cell tumor of bone (GCT) presents with numerous osteoclast-like multinucleated giant cells that are principally responsible for the extensive bone resorption by the tumor. Although the precise etiology of GCT remains uncertain, the accumulation of giant cells is partially due to the high expression of the receptor activator of nuclear factor-κB ligand (RANKL) from the neoplastic stromal cells. Here, we have investigated whether parathyroid hormone-related protein (PTHrP) plays a role in the pathogenesis of GCT. Immunohistochemistry results revealed PTHrP expression in the stromal cells of the tumor, and that its receptor, the parathyroid hormone type 1 receptor (PTH1R), is expressed by both the stromal cells and giant cells. PCR and Western blot analyses confirmed the expression of PTHrP and PTH1R by isolated stromal cells from five patients presenting with GCT. Treatment of GCT stromal cells with varying concentrations of PTHrP (1-34) significantly increased both RANKL gene expression and the number of multinucleated cells formed from RAW 264.7 cells in co-culture experiments, whereas inhibition of PTHrP with a neutralizing antibody decreased RANKL gene expression. These results suggest that PTHrP is expressed within GCT by the stromal cells and can contribute to the abundant RANKL expression and giant cell formation within the tumor.     

地诺单抗通过STAT3信号通路抑制骨巨细胞瘤的初步研究

目的 研究STAT3信号通路及其下游相关分子在地诺单抗治疗骨巨细胞瘤过程中的表达变化及其意义。方法 收集我院2013年1月至2018年12月手术治疗的31例骨巨细胞瘤病人,其中28例未经地诺单抗治疗（对照组）,3例经地诺单抗治疗（研究组）。通过苏木素-伊红（hematoxylin and eosin, HE）染色检测骨巨细胞瘤组织经地诺单抗治疗前后的病理学变化;通过免疫组化法检测研究组和对照组的骨巨细胞瘤组织中RANKL、STAT3及其下游分子Bcl-2、Cyclin D1分子的表达差异;通过TUNEL法检测上述两组石蜡切片组织中肿瘤细胞的凋亡情况。结果 HE染色结果：对照组中骨巨细胞瘤组织主要由肿瘤基质细胞和多核破骨样巨细胞组成;研究组中破骨样巨细胞消失,残留部分细长形肿瘤基质细胞,大量网状纤维组织及编织骨形成并替代肿瘤组织;免疫组化检测结果：RANKL主要表达于肿瘤基质细胞;STAT3主要表达于多核破骨细胞胞浆和肿瘤基质细胞胞膜;Bcl-2主要表达于多核破骨样巨细胞胞浆、散在分布于细胞核;Cyclin D1表达于多核破骨样巨细胞的细胞核中。RANKL、STAT3、Bcl-2和Cyclin D1在对照组肿瘤组织中的阳性表达率分别为70%、53%、77%、73%;研究组肿瘤组织中多核破骨样巨细胞消失,残留的肿瘤基质细胞中RANKL表达量明显减少,未见STAT3、Bcl-2、Cyclin D1分子表达;TUNEL法凋亡结果：对照组中仅有少量的肿瘤细胞凋亡,研究组中可见残留的肿瘤细胞明显凋亡。结论 地诺单抗可能通过抑制STAT3 信号通路抑制多核破骨样巨细胞的形成及促进肿瘤基质细胞凋亡。     

5-Fluoruracil blocked giant cell tumor progression by suppressing osteoclastogenesis through NF-kappaB signals and blocking angiogenesis

Giant cell tumor of bone (GCTB) is a bone destroying tumor comprised of spindle-like stromal cells and monocytes of myeloid lineage that are differentiated into osteoclast-like multinucleated giant cells. Nuclear factor-Kappa B (NF-κB) has been identified to be essential for GCT progression. Herein, we found that 5-Fluorouracil (5-FU), a widely used chemotherapeutics, is a promising anticancer agent for GCT both targeting spindle-like stromal cells and osteoclast giant cells through NF-κB pathway. In this study, in vitro 5-FU not only directly blocked both stromal cell- and RANKL-induced osteoclastogenesis through NF-κB pathway, but also indirectly inhibited osteoclast formation and angiogenesis by suppressing the expression of osteoclast-activating factors including IL-1β, MCP-1 and tumor angiogenesis factor VEGF in stromal cells. In vivo, we found that 5-FU blocked GCT progression through NF-κB pathway by utilizing our chick embryo chorioallantoic membrane (CAM) model. Taken together, our results suggest that 5-FU can inhibit GCT development by suppressing osteoclast formation through NF-κB pathway and blocking angiogenesis, and may serve as a novel agent in the treatment of GCT.     

Histogenetic Characterization of Giant Cell Tumor of Bone

The unpredictable behavior of giant cell tumor (GCT) parallels its controversial histogenesis. Multinucleated giant cells, stromal cells, and CD68⁺ monocytes/macrophages are the three elements that interact in GCT. We compared the ability of stromal cells and normal mesenchymal stromal cells to differentiate into osteoblasts. Stromal cells and mesenchymal cells had similar proliferation rates and lifespans. Although stromal cells expressed early osteogenic markers, they were unable to differentiate into osteoblasts but they did express intracellular adhesion molecule-1, a marker of bone-lining cells. They were unable to form clones in a semisolid medium and unable to promote osteoclast differentiation, although they exerted a strong chemotactic effect on osteoclast precursors. Stromal cells may be either immature proliferating osteogenic elements or specialized osteoblast-like cells that fail to show neoplastic features but can induce the differentiation of osteoclast precursors. They might be secondarily induced to proliferate by a paracrine effect induced by monocyte-macrophages and/or giant cells. The increased number of giant cells in GCT may be secondary to an autocrine circuit mediated by the receptor activator of nuclear factor kB. One or more of the authors (NB) has received funding from the Italian Association for Cancer Research. Each author certifies that his or her institution has approved the human protocol for this investigation, that all investigations were conducted in conformity with ethical principles of research, and that informed consent for participation in the study was obtained.     

Visualization of bisphosphonate-induced caspase-3 activity in apoptotic osteoclasts in vitro

Bisphosphonates inhibit osteoclast-mediated bone resorption by mechanisms that have only recently become clear. Whereas nitrogen-containing bisphosphonates affect osteoclast function by preventing protein prenylation (especially geranylgeranylation), non-nitrogen-containing bisphosphonates have a different molecular mechanism of action. In this study, we demonstrate that nitrogen-containing bisphosphonates (risedronate, alendronate, pamidronate, and zoledronic acid) and non-nitrogen-containing bisphosphonates (clodronate and etidronate) cause apoptosis of rabbit osteoclasts, human osteoclastoma-derived osteoclasts, and human osteoclast-like cells generated in cultures of bone marrow in vitro. Osteoclast apoptosis was shown to involve characteristic morphological changes, loss of mitochondrial membrane potential, and the activation of caspase-3-like proteases capable of cleaving peptide substrates with the sequence DEVD. Caspase-3-like activity could be visualized in unfixed, dying osteoclasts and osteoclast-like cells using a cell-permeable, fluorogenic substrate. Bisphosphonate-induced osteoclast apoptosis was dependent on caspase activation, because apoptosis resulting from alendronate, clodronate, or zoledronic acid treatment was suppressed by zVAD-fmk, a broad-range caspase inhibitor, or by SB-281277, a specific isatin sulfonamide inhibitor of caspase-3/-7. Furthermore, caspase-3 (but not caspase-6 or caspase-7) activity could be detected and quantitated in lysates from purified rabbit osteoclasts, whereas the p17 fragment of active caspase-3 could be detected in human osteoclast-like cells by immunofluorescence staining. Caspase-3, therefore, appears to be the major effector caspase activated in osteoclasts by bisphosphonate treatment. Caspase activation and apoptosis induced by nitrogen-containing bisphosphonates are likely to be the consequence of the loss of geranylgeranylated rather than farnesylated proteins, because the ability to cause apoptosis and caspase activation was mimicked by GGTI-298, a specific inhibitor of protein geranylgeranylation, whereas FTI-277, a specific inhibitor of protein farnesylation, had no effect on apoptosis or caspase activity.     

Bisphosphonates may reduce recurrence in giant cell tumor by inducing apoptosis

Giant cell tumor of bone is an aggressive tumor characterized by extensive bone destruction and high recurrence rates. This tumor consists of stromal cells and hematopoietic cells that interact in an autocrine manner to produce tumoral osteoclastogenesis and bone resorption. This autocrine regulation may be disrupted by novel therapeutic agents. Nonspecific local adjuvant therapies such as phenol or liquid nitrogen have been used in the treatment of giant cell tumor, but specific adjuvant therapies have not been described. The bisphosphonates pamidronate and Zoledronate can induce apoptosis in giant cell tumor culture in a dose-dependent manner. We established giant cell tumor cultures from patients with extensive destruction of bone. One of the four cultures formed osteoclastlike giant cells in vitro after more than six passages without exogenous receptor activator of NF-kappaB ligand or macrophage colony stimulating factor. Annexin V staining, presence of active cleaved form of caspase-3, and disappearance of poly (ADP-ribose) polymerase on Western blotting indicated activation of apoptosis by bisphosphonates in giant cell tumor. These results indicate that topical or systemic use of pamidronate or zoledronate can be a novel adjuvant therapy for giant cell tumor by targeting osteoclastlike giant cells, mononuclear giant cell precursor cells, and the autocrine loop of tumor osteoclastogenesis.     

Giant cell tumor of bone: A basic science perspective

Comprehending the pathogenesis of giant cell tumor of bone (GCT) is of critical importance for developing novel targeted treatments for this locally-aggressive primary bone tumor. GCT is characterized by the presence of large multinucleated osteoclast-like giant cells distributed amongst mononuclear spindle-like stromal cells and other monocytes. The giant cells are principally responsible for the extensive bone resorption by the tumor. However, the spindle-like stromal cells chiefly direct the pathology of the tumor by recruiting monocytes and promoting their fusion into giant cells. The stromal cells also enhance the resorptive ability of the giant cells. This review encompasses many of the attributes of GCT, including the process of giant cell formation and the mechanisms of bone resorption. The significance of the receptor activator of nuclear factor-κB ligand (RANKL) in the development of GCT and the importance of proteases, including numerous matrix metalloproteinases, are highlighted. The mesenchymal lineage of the stromal cells and the origin of the hematopoietic monocytes are also discussed. Several aspects of GCT that require further understanding, including the etiology of the tumor, the mechanisms of metastases, and the development of an appropriate animal model, are also considered. By exploring the current status of GCT research, this review accentuates the significant progress made in understanding the biology of the tumor, and discusses important areas for future investigation.     

Expression of osteoclast differentiation signals by stromal elements of giant cell tumors.

The mechanisms by which primary tumors of the bone cause bone destruction have not been elucidated. Unlike most other lytic bone tumors, osteoclastomas, otherwise known as giant cell tumors (GCT), contain osteoclast-like cells within the tumor stroma. A new member of the TNF-ligand superfamily member, osteoclast differentiation factor (ODF/OPGL/RANKL/TRANCE), was recently identified. ODF was shown to directly stimulate osteoclastogenesis, in the presence of M-CSF. In this study, the expression of ODF was examined in a number of tumor samples associated with bone lysis in vivo. In addition, we investigated expression of the ODF receptor on osteoclast precursors, RANK, as well as the ODF inhibitor osteoprotegerin (OPG), and another TNF-ligand superfamily member, TRAIL, previously shown to abrogate the inhibitory effects of OPG. We report here the novel finding that GCT stromal cells contain abundant ODF mRNA, whereas the giant cell population exclusively expresses RANK mRNA. These results are consistent with the osteoclast-mediated bone destruction by these tumors. We also report the expression of OPG and TRAIL mRNA in GCT samples. A comparison with other lytic and nonlytic tumors of bone showed that GCT express more ODF and TRAIL mRNA relative to OPG mRNA. In addition, GCT were found to express a number of cytokines previously reported to play central roles in osteoclastogenesis, namely, IL-1, -6, -11, -17, as well as TNF-alpha. Importantly, GCT were also found to express high levels of M-CSF mRNA, a cytokine shown to be an essential cofactor of ODF, and a survival factor for mature and developing osteoclasts. Furthermore, expression of these molecules by stromal cells isolated from GCT continued in vitro. Thus GCT constitutively express all of the signals that are currently understood to be necessary for the differentiation of osteoclasts from precursor cells.     

Bisphosphonates reduce local recurrence in extremity giant cell tumor of bone: a case-control study

BACKGROUND: Giant cell tumor (GCT) of bone is a benign but locally aggressive tumor that is characterized by the presence of mononuclear stromal cells and multinucleated giant cells. Although topical adjuvants have been used in the past, local recurrence following intralesional excision of GCT of bone continues to remain a problem. The use of bisphosphonates as an anti-osteoclastic agent in the management of osteolytic bone metastases is well accepted. Furthermore in vitro studies have shown that bisphosphonates also induce apoptosis in GCT stromal cells. Therefore our clinical study aims to investigate whether the administration of bisphosphonate as an adjuvant can further decrease local recurrence following the surgical treatment of GCT of bone. METHOD: A retrospective case-control study was performed between 1988 and 2004. Forty-four patients with histological diagnosed appendicular GCT were included. Intralesional curettage or wide excision of the lesions was followed with cementation or biological reconstruction. Additional intravenous and oral bisphosphonates were given peri-operatively to 24 patients who were treated between 1998 and 2004. The average follow-up of the control group was 115 months while that of the treatment group was 48 months. RESULTS: In the bisphosphonate treated group, 1 of 24 patients (4.2%) developed local recurrence. In the control group, 6 of 20 patients (30%) developed local recurrence. The difference in the recurrence rate was statistically significant between the bisphosphonate treatment group and the control group (Log Rank test p=0.056). The effect of reduction of local recurrence was significant in patients with stage III diseases. Patients treated with bisphosphonate did not report any untoward effects. CONCLUSION: Clinical use of bisphosphonates as an adjuvant therapy for giant cell tumor of bone demonstrated a lower local recurrence rate. The clinical response seems to be more promising in stage III diseases.     

Giant cell tumour of the first metatarsal: a case report

The classical giant cell tumour (GCT) is a solitary lesion characterised by benign-appearing osteoclast-like giant cells and stromal cells that originate within the epiphysis of adults. Long tubular bones (75–90%) are frequent sites of involvement and make up 5% of all primary bone tumours. The foot is a rare site for GCT; less than 50 cases of GCT in foot bones have been reported in the literature. It is easy to misdiagnose a lytic lesion in the foot for commoner disorders. We report a case of GCT of the first metatarsal and highlight the importance of early and proper diagnosis.     

Aminobisphosphonates Cause Osteoblast Apoptosis and Inhibit Bone Nodule Formation In Vitro

Bisphosphonates are widely used for the treatment of bone diseases associated with increased osteoclastic bone resorption. Bisphosphonates are known to inhibit biochemical markers of bone formation in vivo, but it is unclear to what extent this is a consequence of osteoclast inhibition or a direct inhibitory effect on cells of the osteoblast lineage. In order to investigate this issue, we studied the effects of various bisphosphonates on osteoblast growth and differentiation in vitro. The aminobisphosphonates pamidronate and alendronate inhibited osteoblast growth, caused osteoblast apoptosis, and inhibited protein prenylation in osteoblasts in a dose-dependent manner over the concentration range 20-100 microM. Further studies showed that alendronate in a dose of 0.1 mg/kg inhibited protein prenylation in calvarial osteoblasts in vivo, indicating that alendronate can be taken up by osteoblasts in sufficient amounts to inhibit protein prenylation at clinically relevant doses. Pamidronate and alendronate inhibited bone nodule formation at concentrations 10-fold lower than those required to inhibit osteoblast growth. These effects were not observed with non-nitrogen-containing bisphosphonates or with other inhibitors of protein prenylation and were only partially reversed by cotreatment with a fourfold molar excess of ss-glycerol phosphate. We conclude that aminobisphosphonates cause osteoblast apoptosis in vitro at micromolar concentrations and inhibit osteoblast differentiation at nanomolar concentrations by mechanisms that are independent of effects on protein prenylation and may be due in part to inhibition of mineralization. While these results need to be interpreted with caution because of uncertainty about the concentrations of bisphosphonates that osteoblasts are exposed to in vivo, our studies clearly demonstrate that bisphosphonates exert strong inhibitory effects on cells of the osteoblast lineage at similar concentrations to those that cause osteoclast inhibition. This raises the possibility that inhibition of bone formation by bisphosphonates may be due in part to a direct inhibitory effect on cells of the osteoblast lineage.     

Recruitment of osteoclast precursors by stromal cell derived factor-1 (SDF-1) in giant cell tumor of bone.

Giant cell tumor (GCT) of bone is a unique bone lesion that is characterized by an excessive number of multinucleated osteoclasts. GCT consists of neoplastic stromal cells, multinucleated osteoclasts and their precursors, thus serving as a naturally occurring human disease model for the study of osteoclastogenesis. It still remains unclear how stromal cells of GCT recruit osteoclast precursors. In the present study, we characterized the cellular components of GCT and confirmed the presence of CD14(+)-monocytes/CD68(+)-macrophages and CD34(+)-hematopoetic stem cells that express CXCR4, a specific receptor for SDF-1; SDF-1 gene expression and presence of SDF-1 protein were confirmed by real time RT-PCR, in situ hybridization, and immunohistochemistry in the GCT tissue and cultured cells. SDF-1 was present at 25-50 ng/ml in the conditioned media from the GCT cultures, which is in the range of physiological chemotactic concentration. Migration of osteoclast precursors was 2.5-fold higher in response to GCT conditioned media compared to the control media; and migration was inhibited by an average of 36% with anti-SDF-1 neutralizing antibody or competing recombinant SDF-1. These results suggest that SDF-1 is one of the significant chemoattractant factors involved in the recruitment of hematopoietic osteoclast precursor cells during tumor-induced osteoclastogenesis.     

Pamidronate,farnesyl transferase,and geranylgeranyl transferase‐I inhibitors affects cell proliferation,apoptosis, and OPG/RANKL mRNA expression in stromal cells of giant cell tumor of bone

Giant cell tumor (GCT) is the most common nonmalignant primary bone tumor reported in Hong Kong. It usually affects young adults between the ages of 20 and 40. This tumor is well known for its potential to recur following treatment. To date no effective adjuvant therapy exists for GCT. Our project aimed to study the effects of pamidronate (PAM), farnesyl transferase inhibitor (FTI‐277), geranylgeranyl transferase inhibitor (GGTI‐298), and their combinations on GCT stromal cells (SC). Individual treatment with PAM, FTI‐277, and GGTI‐298, inhibited the cell viability and proliferation of GCT SC in a dose‐dependent way. Combination of FTI‐277 with GGTI‐298 caused synergistic effects in reducing cell viability, and its combination index was 0.49, indicating a strong synergism. Moreover, the combination of FTI‐277 with GGTI‐298 synergistically enhanced cell apoptosis and activated caspase‐3/7, ‐8, and ‐9 activities. PAM induced cell‐cycle arrest at the S‐phase. The combination of PAM with GGTI‐298 significantly increased OPG/RANKL mRNA ratio and activated caspase‐3/7 activity. Our findings support that the combination of bisphosphonates with GGTIs or FTIs with GGTIs may be used as potential adjuvants in the treatment of GCT of bone. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 29:403–413, 2011     

Osteoprotegerin inhibits osteoclast formation and bone resorbing activity in giant cell tumors of bone

Osteolysis is a common complication of tumors that arise in, or metastasize to, bone. The recent discovery of key regulators of osteoclast formation and activity, including receptor activator of nuclear factor of kappaB ligand (RANKL), RANK, and osteoprotegerin (OPG), may facilitate new treatment regimes for certain tumors associated with excessive bone loss. We recently showed that the stromal cells of osteolytic giant cell tumors (GCT) of bone express high levels of mRNA encoding RANKL, relative to mRNA for the RANKL antagonist, OPG, compared with the expression patterns of other lytic and nonlytic bone tumors. In this study, we found that expression of RANKL and OPG mRNA continued by the stromal element of these tumors in a constitutive manner for at least 9 days in the absence of giant cells. Immunostaining of unfractionated GCT cultured in vitro revealed punctate cytoplasmic/membranous staining for RANKL and both cytoplasmic and extracellular matrix staining for OPG in stromal cells. Giant cells (osteoclasts) were negative for RANKL staining, but stained brightly for cytoplasmic OPG protein. We also investigated the functional relevance of these molecules for GCT osteolysis by adding recombinant OPG and RANKL to cultured GCT cells. We found that OPG treatment potently and dose-dependently inhibited resorption of bone slices by GCT, and could also inhibit the formation of multinucleated osteoclasts from precursors within the GCT. These effects of OPG were reversed by stoichiometric concentrations of exogenous RANKL. These data indicate that both the processes of osteoclast formation and activation in GCT are promoted by RANKL. Therefore, GCT represent a paradigm for the direct stimulation of osteoclast formation and activity by tumor stromal cells, in contrast to the mechanisms described for osteolytic breast tumors and multiple myeloma. The demonstration of these relationships is important in developing approaches to limit tumor-induced osteolysis.     

Ultrastructural and cytobiological studies on possible interactions between PTHrP-secreting tumor cells,stromal cells,and bone cells

Parathyroid hormone-related peptide (PTHrP) induces pathological bone resorption in an endocrine manner, resulting in hypercalcemia of malignancy. However, the histopathological aspect of the action of PTHrP secreted by tumor cells on bone resorption has not well been documented. Therefore, we studied cell–cell interactions between bone cells, stromal cells, and PTHrP-secreting tumor cells (EC-GI-10) morphologically. Tumor cells injected subcutaneously into the parietal region formed a tumor mass, invading the bone marrow. The tumor mass was surrounded by a membrane structure consisting of stromal cells. These stromal cells were positive for alkaline phosphatase (ALPase). Tartrate-resistant acid phosphatase (TRAPase)-positive osteoclasts were localized close to the ALPase-positive cells, and numerous osteoclasts were observed on the neighboring bone surfaces. PTHrP, vascular endothelial growth factor (VEGF), and matrix metalloproteinase (MMP)-9 were detected in the tumor cells. Using RT-PCR, expression of interleukin (IL)-1, IL-1, and PTHrP, which are strong bone resorption factors, was detected in the tumor cells. Some ALPase-positive cells localizing on the neighboring bone surfaces and endothelial cells revealed PTH/PTHrP receptor immunoreactivity. Ultrastructurally, numerous blood vessels were observed between the tumor nests and the stromal cells. The nests were surrounded by a basement membrane, but it was discontinuous, therefore permitting direct contact between the tumor cells and the stromal cells. These results indicate that PTHrP secreted by tumor cells appears to stimulate osteoclast differentiation and bone resorption in a paracrine manner through PTH/PTHrP receptor-immunopositive cells. IL-1, IL-1, VEGF, and MMP-9 may also be involved in facilitating osteoclast formation and the subsequent bone resorption.     

Expression of preosteoblast markers and Cbfa-1 and Osterix gene transcripts in stromal tumour cells of giant cell tumour of bone

In giant cell tumour of bone (GCT), mononuclear stromal cells, which represent the neoplastic component of this lesion, regulate the formation of multinucleated osteoclast-like giant cells which are the characteristic hallmark of this tumour. However, the origin of stromal tumour cells has not yet been clearly defined. In this study, we evaluated several osteoblast markers including collagen type I, bone sialoprotein (BSP), osteonectin and osteocalcin in GCT using immunohistochemical techniques. Amongst the 13 GCT specimens and 7 GCT stromal cell (GCTSC) cultures studied, majority of the GCTSC synthesized type I collagen, BSP and osteonectin proteins but did not produce the differentiated osteoblast marker, osteocalcin. We further examined the regulation of several important osteogenic genes such as Cbfa-1, osterix and osteocalcin, and regulation of ALP activity in GCTSC in culture by bone morphogenetic protein 2 (BMP-2). Real-time PCR analysis indicated that Cbfa-1, osterix and osteocalcin mRNA were present in primary cultures of GCTSC. The addition of BMP-2 upregulated Cbfa-1 and osterix gene expression within 12 h and the enhancement was still observed at 24 h. ALP activity was minimal in untreated GCTSC in cultures. The number of ALP-positive GCTSC was significantly increased following treatment with BMP-2 or combinations with beta-glycerophosphate and ascorbic acid. In contrast, BMP enhancement of osterix mRNA level and ALP activity was also seen in SaOS2 osteoblast-like cells, but not in the primary culture of normal human skin fibroblasts. In summary, our data suggest that GCT stromal tumour cells may have an osteoblastic lineage and retain the ability to differentiate into osteoblasts.     

Inhibition of bone resorption by alendronate and risedronate does not require osteoclast apoptosis

Bisphosphonate inhibition of bone resorption was proposed to be due to osteoclast apoptosis. We tested this hypothesis for both the N-containing bisphosphonates alendronate and risedronate, which inhibit farnesyldiphosphate synthase and thus protein isoprenylation, and for clodronate and etidronate, which are metabolized to adenosine triphosphate (ATP) analogs. We found, in dose-response studies, that alendronate and risedronate inhibit bone resorption (in pit assays) at doses tenfold lower than those reducing osteoclast number. At an N-bisphosphonate dose that inhibited resorption and induced apoptosis, the antiapoptotic caspase inhibitor, Z-VAD-FMK, maintained osteoclast (Oc) number but did not prevent inhibition of resorption. Furthermore, when cells were treated with either alendronate alone or in combination with Z-VAD-FMK for 24 or 48 h, subsequent addition of geranylgeraniol, which restores geranylgeranylation, returned bone resorption to control levels. On the other hand, Z-VAD-FMK did block etidronate and clodronate inhibition of resorption. Moreover, in cells treated with etidronate, but not alendronate or risedronate, Z-VAD-FMK also prevented actin disruption, an early sign of osteoclast inhibition by bisphosphonates. These observations indicate that, whereas induction of apoptosis plays a major role in etidronate and clodronate inhibition of resorption, alendronate and risedronate suppression of bone resorption is independent of their effects on apoptosis.     

Role of intravenous zoledronic acid in management of giant cell tumor of bone- A prospective,randomized, clinical,radiological and electron microscopic analysis

BackgroundThe primary treatment of Giant cell tumor of bone is surgical management. Bisphosphonates are antiresorptive drugs which inhibit osteoclast mediated bone resorption and shown to have inhibitory effect on various tumors. The present study aims to establish clinical, ultrastructural and radiological response of intravenous zoledronic acid on giant cell tumor of bone.MethodologyDesign - Prospective randomized controlled study. A group of 30 patients of GCT bone were randomized into two equal groups. Patients in control group did not receive any adjuvant therapy before surgery. Patients in bisphosphonate group received three doses of intravenous zoledronic acid at four weeks interval prior to definitive surgery. The evaluation was done based on size of swelling, VAS score, plain radiograph, MRI and histopathological and Transmission electron microscopic examination findings.ResultsSignificant reduction in VAS score (from mean 5.33 to 1.8), increased mineralization particularly at periphery of lesion in plain radiograph, statistically significant increase in mean apoptotic index, P value < 0.0001 (mean 41.46 in bisphosphonate group and 6.06 in control group) was noted in bisphosphonate group. No significant change in tumor volume is noted in MRI. No significant side effects were noted.DiscussionOne distinctive feature of pathogenesis of GCT bone is osteoclastogenesis which causes extensive bone destruction. Use of intravenous Zoledronic acid counteracts this bone destruction. Further, possible antiangiogenic effect of intravenous bisphosphonates inhibits tumor growth and provides symptomatic improvement.ConclusionIV Zoledronic acid alleviates pain, produce sclerosis and induce apoptosis hence decrease the rate of tumor progression and decrease the rate of local bone destruction, hence they are useful adjuvant to surgery in GCT.     

Protein geranylgeranylation is required for osteoclast formation, function, and survival: inhibition by bisphosphonates and GGTI-298.

Bisphosphonates are the important class of antiresorptive drugs used in the treatment of metabolic bone diseases. Although their molecular mechanism of action has not been fully elucidated, recent studies have shown that the nitrogen-containing bisphosphonates can inhibit protein prenylation in macrophages in vitro. In this study, we show that the nitrogen-containing bisphosphonates risedronate, zoledronate, ibandronate, alendronate, and pamidronate (but not the non nitrogen-containing bisphosphonates clodronate, etidronate, and tiludronate) prevent the incorporation of [14C]mevalonate into prenylated (farnesylated and geranylgeranylated) proteins in purified rabbit osteoclasts. The inhibitory effect of nitrogen-containing bisphosphonates on bone resorption is likely to result largely from the loss of geranylgeranylated proteins rather than loss of farnesylated proteins in osteoclasts, because concentrations of GGTI-298 (a specific inhibitor of geranylgeranyl transferase I) that inhibited protein geranylgeranylation in purified rabbit osteoclasts prevented osteoclast formation in murine bone marrow cultures, disrupted the osteoclast cytoskeleton, inhibited bone resorption, and induced apoptosis in isolated chick and rabbit osteoclasts in vitro. By contrast, concentrations of FTI-277 (a specific inhibitor of farnesyl transferase) that prevented protein farnesylation in purified rabbit osteoclasts had little effect on osteoclast morphology or apoptosis and did not inhibit bone resorption. These results therefore show the molecular mechanism of action of nitrogen-containing bisphosphonate drugs in osteoclasts and highlight the fundamental importance of geranylgeranylated proteins in osteoclast formation and function.