Mice lacking 5-lipoxygenase have increased cortical bone thickness.
Explore the source record for details and available documents.
Animals↗
Biomedical subjects
B F Boyce
Publications and source records attributed to B F Boyce.
Apoptosis in bone physiology and disease.
Explore the source record for details and available documents.
Apoptosis↗
Evidence for a causal role of parathyroid hormone-related protein in the pathogenesis of human breast cancer-mediated osteolysis.
Breast cancer almost invariably metastasizes to bone in patients with advanced disease and causes local osteolysis. Much of the morbidity of advanced breast cancer is a consequence of this process. Despite the importance of the problem, little is known of the pathophysiology of local osteolysis in the skeleton or its prevention and treatment. Observations in patients with bone metastases suggest that breast cancer cells in bone express parathyroid hormone-related protein (PTHrP) more frequently than in soft tissue sites of metastasis or in the primary tumor. Thus, the role of PTHrP in the causation of breast cancer metastases in bone was examined using human breast cancer cell lines. Four of eight established human breast cancer cell lines expressed PTHrP and one of these cell lines, MDA-MB-231, was studied in detail using an in vivo model of osteolytic metastases. Mice inoculated with MDA-MB-231 cells developed osteolytic bone metastasis without hypercalcemia or increased plasma PTHrP concentrations. PTHrP concentrations in bone marrow plasma from femurs affected with osteolytic lesions were increased 2.5-fold over corresponding plasma PTHrP concentrations. In a separate experiment, mice were treated with either a monoclonal antibody directed against PTHrP(1-34), control IgG, or nothing before tumor inoculation with MDA-MB-231 and twice per week for 26 d. Total area of osteolytic lesions was significantly lower in mice treated with PTHrP antibodies compared with mice receiving control IgG or no treatment. Histomorphometric analysis of bone revealed decreased osteoclast number per millimeter of tumor/bone interface and increased bone area, as well as decreased tumor area, in tumor-bearing animals treated with PTHrP antibodies compared with respective controls. These results indicate that tumor-produced PTHrP can cause local bone destruction in breast cancer metastatic to bone, even in the absence of hypercalcemia or increased circulating plasma concentrations of PTHrP. Thus, PTHrP may have an important pathogenetic role in the establishment of osteolytic bone lesions in breast cancer. Neutralizing antibodies to PTHrP may reduce the development of destructive bone lesions as well as the growth of tumor cells in bone.
Animals↗
Metastatic prostate cancer in a transgenic mouse.
We have previously reported the development of a transgenic mouse model for prostate cancer derived from PB-Tag transgenic line 8247, henceforth designated the TRAMP (transgenic adenocarcinoma mouse prostate) model. We now describe the temporal and spatial consequences of transgene expression and report the identification and characterization of metastatic disease in the TRAMP model. TRAMP mice characteristically express the T antigen oncoprotein by 8 weeks of age and develop distinct pathology in the epithelium of the dorsolateral prostate by 10 weeks of age. Distant site metastases can be detected as early as 12 weeks of age. The common sites of metastases are the periaortic lymph nodes and lungs, with occasional metastases to the kidney, adrenal gland, and bone. By 28 weeks of age, 100% harbor metastatic prostate cancer in the lymph nodes or lungs. We have also demonstrated the loss of normal E-cadherin expression, as observed in human prostate cancer, as primary tumors become less differentiated and metastasize. The TRAMP model provides a consistent source of primary and metastatic tumors for histopathobiological and molecular analysis to further define the earliest molecular events involved in the genesis, progression, and metastasis of prostate cancer.
Adenocarcinoma↗
A new model for the regulation of bone resorption, with particular reference to the effects of bisphosphonates.
Explore the source record for details and available documents.
Animals↗
Leukotriene B4 stimulates osteoclastic bone resorption both in vitro and in vivo.
Upon activation, the enzyme 5-lipoxygenase converts arachidonic acid into principally three products, the peptidoleukotrienes, 5-hydroperoxyeicosatetraenoic acid (5-HETE) or the leukotriene B4. We have shown that the peptido-leukotrienes (known as LTC4, LTD4, or LTE4) and 5-HETE induce osteoclastic bone resorption and that receptors for LTD4 are present on isolated avian osteoclast-like cells. Here, we show the effects of the third metabolic product of the 5-lipoxygenase (5-LO) pathway of arachidonic acid metabolism, the leukotriene LTB4, on osteoclastic bone resorption both in vivo and in vitro. Because LTB4 production is increased in a number of inflammatory conditions, it may be an important contributor to the bone loss which occurs in these disorders. LTB4 increased osteoclastic bone resorption in vivo following local administration over the calvariae of normal mice and in vitro in organ cultures of neonatal mouse calvariae. When LTB4 was injected over the calvaria of mice, there was a significant increase in bone resorption, osteoclast numbers, and eroded surfaces. LTB4 also increased the formation of resorption lacunae by isolated neonatal rat osteoclasts. Greater potency was observed with LTB4 compared with the peptido-leukotriene LTD4. This is in contrast to prostaglandins of the E series, which are reported to inhibit isolated osteoclasts. Experiments using marrow cultures suggest that LTB4 stimulates bone resorption in part by enhancing the formation of osteoclasts.
Analysis of Variance↗
Estrogen promotes apoptosis of murine osteoclasts mediated by TGF-beta.
Postmenopausal osteoporosis, the most common bone disease in the developed world, is associated with estrogen deficiency. This deficiency induces increased generation and activity of osteoclasts, which perforate bone trabeculae, thus reducing their strength and increasing fracture risk. Estrogen replacement prevents these effects, indicating that estrogen negatively regulates osteoclast formation and function, but how it does this is unclear. Because functional osteoclast life span and thus the amount of bone that osteoclasts resorb could also be enhanced following estrogen deficiency, and since sex steroids regulate apoptosis in other target tissues, we investigated whether estrogen may affect osteoclast function by promoting apoptosis. 17 beta-Estradiol promoted apoptosis of murine osteoclasts in vitro and in vivo by two- to threefold. Tamoxifen, which has estrogenic effects on bone resorption, and transforming growth factor-beta 1 (TGF-beta), whose production by osteoblasts is increased by estrogen, had similar effects in vitro. Anti-TGF-beta antibody inhibited TGF-beta-, estrogen- and tamoxifen-induced osteoclast apoptosis, indicating that TGF-beta might mediate this effect. These findings suggest that estrogen may prevent excessive bone loss before and after the menopause by limiting osteoclast life span through promotion of apoptosis. The development of analogues to promote this mechanism specifically could be a useful and novel therapeutic approach to prevent postmenopausal osteoporosis.
Animals↗
Bisphosphonate risedronate reduces metastatic human breast cancer burden in bone in nude mice.
Human breast cancer frequently metastasizes to the skeleton to cause osteolysis and subsequent pain, pathological fracture, and hypercalcemia. Because bone continuously releases growth factors stored in bone matrix by bone resorption during physiological remodeling and, thus, possibly provides a favorable microenvironment for metastatic breast cancer cells to proliferate, inhibitors of bone resorption used either prophylactically or in patients with established disease, therefore, would seem likely to be useful adjuvant therapy in patients with breast cancer. However, the parameters for monitoring progressive osteolytic bone disease in humans are imprecise. We examined the effects of the third generation bisphosphonate, risedronate, which is a specific inhibitor of osteoclastic bone resorption, in a bone metastasis model in nude mice in which intracardiac injection of the human breast cancer cell line MDA-231 leads to osteolytic bone metastases. Risedronate (4 micrograms/animal/day) was given s.c. to animals (a) after radiologically small but defined osteolytic metastases were observed; (b) simultaneously with MDA-231 cell inoculation through the entire experimental period; or (c) by short-term prophylactic administration before inoculation of MDA-231 cells. In all experiments, risedronate either slowed progression or inhibited the development of bone metastases assessed radiographically. Furthermore, mice treated continuously with risedronate showed significantly longer survival than did control mice. Histomorphometrical analysis revealed that osteoclast numbers were diminished at metastatic tumor sites. Unexpectedly, there was also a marked decrease in tumor burden in bone in risedronate-treated animals. In contrast, the growth of metastatic breast cancer in soft tissues surrounding bones was not affected by risedronate. Moreover, risedronate had no effects on the local growth of s.c. implanted MDA-231 breast cancers in nude mice or on MDA-231 cell proliferation in culture. These data demonstrate that risedronate decreases metastatic MDA-231 breast cancer burden selectively in bone, as well as suppresses progression of established osteolytic lesions and prevents the development of new osteolytic lesions; thus, the data suggest that inhibition of osteoclastic bone resorption may be a useful adjunctive therapy for the treatment of cancers that have colonized in bone.
Animals↗
Suramin suppresses hypercalcemia and osteoclastic bone resorption in nude mice bearing a human squamous cancer.
Suramin is a polyanionic agent which has been found to be an effective antineoplastic agent against various human tumors including adrenal, renal and prostatic cancer, and osteosarcoma. Recently, suramin has been shown to inhibit bone resorption in organ cultures of mouse calvarial bones. In the present study, we examined the effects of suramin on increased osteoclastic bone resorption and hypercalcemia in nude mice bearing a human oral squamous carcinoma. Suramin (1 mg/mouse/injection) was administered i.p. three times a week for the first 2 weeks and then once weekly for the next 6 weeks. Blood ionized calcium levels in the suramin-treated cancer-bearing group were significantly lower than those in the untreated cancer-bearing group. Histological and histomorphometrical examination of bones of these animals showed a significant decrease in osteoclast numbers in the suramin-treated cancer-bearing animals. Suramin at a dose of 0.1 mg/mouse/injection was ineffective and 2 mg/mouse/injection was toxic, confirming its narrow effective dose. Suramin showed no effects on the growth of this squamous cancer. However, suramin markedly inhibited in vivo growth of a rat prostatic adenocarcinoma. In mouse marrow cultures, suramin decreased osteoclast-like cell formation in a dose-dependent manner. Furthermore, suramin also inhibited bone resorption in organ cultures of fetal rat long bones and resorption pit formation by isolated mature rat osteoclasts. These results show that suramin is an effective inhibitor of osteoclastic bone resorption in vitro and in vivo and suggest that suramin may be a useful agent in prevention and treatment of cancer-induced hypercalcemia. However, our results also suggest that for this indication suramin has a confined range of effective dose.
Adenocarcinoma↗
Use of an in vivo model to determine the effects of interleukin-1 on cells at different stages in the osteoclast lineage.
In vitro model systems have been used extensively to study factors that affect osteoclast formation and to identify osteoclast precursors. However, in vitro systems do not examine the entire process of osteoclast differentiation simultaneously and lack accessory cells normally present in vivo. Additionally, the role that metabolism of the factor may play on its osteotropic activity in vivo is not addressed by these culture systems. Therefore, we have developed an in vivo model that permits us to examine simultaneously the effects of osteotropic factors on three distinct stages of osteoclast differentiation: (1) multipotent osteoclast precursors, the granulocyte-macrophage colony-forming unit (CFU-GM); (2) more differentiated marrow mononuclear osteoclast precursors; and (3) mature osteoclasts already present on bone surfaces. In the current study, we used interleukin-1 (IL-1) as a prototypic osteotropic factor to test the utility of this system to delineate the cellular mechanisms responsible for enhanced osteoclast activity stimulated by this cytokine. IL-1 induced hypercalcemia and enhanced the growth and differentiation of CFU-GM, increased the number of more committed mononuclear osteoclast precursors, and stimulated mature osteoclasts to resorb bone. These data demonstrate that this simple in vivo model permits the easy delineation of the stages of osteoclast development, in which osteotropic factors act to enhance bone turnover, and may be useful in understanding the mechanism of action of antiresorptive agents.
Analysis of Variance↗
Bisphosphonates promote apoptosis in murine osteoclasts in vitro and in vivo.
Bisphosphonates inhibit bone resorption and are therapeutically effective in diseases of increased bone turnover, such as Paget's disease and hypercalcemia of malignancy. The mechanisms by which they act remain unclear. Proposed mechanisms include inhibition of osteoclast formation from precursors and inhibitory or toxic effect on mature osteoclasts. We have developed a new in vitro model to study osteoclast survival and in this paper present in vitro and in vivo evidence that may explain both the observed reduction in osteoclast numbers and in bone resorption by mature osteoclasts, namely that bisphosphonates induce programmed cell death (apoptosis). Three bisphosphonates (risedronate, pamidronate, and clodronate) caused a 4- to 24-fold increase in the proportion of osteoclasts showing the characteristic morphology of apoptosis in vitro. This observation was confirmed in vivo in normal mice, in mice with increased bone resorption, and in nude mice with osteolytic cancer metastases, with similar-fold increases to those observed in vitro. Of the three compounds, risedronate, the most potent inhibitor of bone resorption in vivo, was the strongest inducer of osteoclast apoptosis in vitro. Osteoclast apoptosis may therefore be a major mechanism whereby bisphosphonates reduce osteoclast numbers and activity, and induction of apoptosis could be a therapeutic goal for new antiosteoclast drugs.
Animals↗
The role of cadherin in the generation of multinucleated osteoclasts from mononuclear precursors in murine marrow.
A critical step in bone resorption is the fusion of mononuclear osteoclast precursors to form multinucleated osteoclasts. However, little is known of the molecular mechanisms that are responsible for this important process. Since the expression of proteins in the cadherin family of homophilic calcium-dependent cell adhesion molecules is involved in the fusion process for certain other cells, we examined their role in osteoclast formation. Immunohistochemical examination of human and mouse bone using monoclonal antibodies to human and mouse E-cadherin clearly demonstrated positive staining in osteoclasts. N- and P-cadherin were not detected. In cultures of murine marrow mononuclear cells in which osteoclasts form by cell fusion, E-cadherin expression determined by Western blotting reached the highest levels as fusion was taking place. Expression of E-cadherin gene fragment was also detected in the marrow cultures by polymerase chain reaction. To study the functional role of E-cadherin expression in osteoclastic differentiation, neutralizing monoclonal antibodies were examined for their effects on osteoclast formation. The antibodies decreased the number of tartrate-resistant acid phosphatase (a marker of murine osteoclast)-positive multinucleated cell (TRAP-positive MNC) by inhibiting the fusion of mononuclear osteoclast precursors, but not proliferation of these cells or their attachment to plastic dish surfaces. This inhibitory effect was reversible. Furthermore, synthetic peptides containing the cell adhesion recognition sequence of cadherins also decreased TRAP-positive MNC formation. The antibodies and peptides inhibited not only osteoclast formation but also bone resorption. Antibodies to other types of cadherins and control rat IgG had no effects in these culture systems. Our findings suggest that E-cadherin expression may be involved in fusion (differentiation) of hemopoietic osteoclast precursors into mature multinucleated osteoclasts.
Amino Acid Sequence↗
Interleukin-6 enhances hypercalcemia and bone resorption mediated by parathyroid hormone-related protein in vivo.
Tumors frequently induce the multifunctional cytokine IL-6, which has been linked to several paraneoplastic syndromes, most notably cachexia. IL-6 stimulates osteoclast formation, causes mild hypercalcemia, and is produced by bone cells in vitro upon exposure to systemic hormones. Since IL-6 is produced together with parathyroid hormone-related protein (PTH-rP) in some patients with cancer, we tested the hypothesis that production of IL-6 potentiates the effects of PTH-rP on Ca2+ homeostasis and osteoclastic bone resorption and examined potential mechanisms for these interactions in vivo. Chinese hamster ovarian (CHO) cells stably transfected with cDNAs for IL-6 (CHO/IL-6) and PTH-rP sense (CHO/PTH-rP) or antisense (CHO/PTH-rP AS) were inoculated intramuscularly into nude mice. Experimental groups included CHO/IL-6 plus CHO/PTH-rP; CHO/IL-6 plus CHO/PTH-rP AS; CHO/IL-6 alone; and CHO/PTH-rP alone. Blood ionized Ca2+ was measured on days 0, 7, 10, 12, and 13. Three different developmental stages in the osteoclast lineage were examined at day 13: the early multipotential precursor, granulocyte macrophage colony-forming units (CFU-GM); more mature mononuclear osteoclast precursors, assessed by their capacity to form tartrate-resistant acid phosphatase-positive multinucleated cells in marrow cultures; and mature osteoclasts, assessed by histomorphometry. IL-6 increased CFU-GM but not bone resorption or Ca2+. In contrast, PTH-rP induced hypercalcemia and bone resorption and increased multinucleated osteoclasts and more mature precursors cells, but not CFU-GM. However, mice treated with both IL-6 and PTH-rP had very marked hypercalcemia and osteoclastosis as well as an increase in the number of both CFU-GM and mature osteoclast precursors. These data demonstrate that IL-6 enhances PTH-rP-mediated hypercalcemia and bone resorption, most likely by increasing the pool of early osteoclast precursors that in turn can differentiate to mature osteoclasts. We conclude that IL-6 stimulatory effects on osteoclast precursors may enhance the effects of other bone resorption factors that act at later stages in the osteoclast lineage.
Animals↗
Regulation of interleukin-6, osteoclastogenesis, and bone mass by androgens. The role of the androgen receptor.
Interleukin-6 is an essential mediator of the bone loss caused by loss of estrogens. Because loss of androgens also causes bone loss, we have examined whether the IL-6 gene is regulated by androgens, and whether IL-6 plays a role in the bone loss caused by androgen deficiency. Both testosterone and dihydrotestosterone inhibited IL-6 production by murine bone marrow-derived stromal cells. In addition, testosterone, dihydrotestosterone, and adrenal androgens inhibited the expression of a chloramphenicol acetyl transferase reporter plasmid driven by the human IL-6 promoter in HeLa cells cotransfected with an androgen receptor expression plasmid; however, these steroids were ineffective when the cells were cotransfected with an estrogen receptor expression plasmid. In accordance with the in vitro findings, orchidectomy in mice caused an increase in the replication of osteoclast progenitors in the bone marrow which could be prevented by androgen replacement or administration of an IL-6 neutralizing antibody. Moreover, bone histomorphometric analysis of trabecular bone revealed that, in contrast to IL-6 sufficient mice which exhibited increased osteoclast numbers and bone loss following orchidectomy, IL-6 deficient mice (generated by targeted gene disruption) did not. This evidence demonstrates that male sex steroids, acting through the androgen-specific receptor, inhibit the expression of the IL-6 gene; and that IL-6 mediates the upregulation of osteoclastogenesis and therefore the bone loss caused by androgen deficiency, as it does in estrogen deficiency.
Animals↗
Targeting simian virus 40 T antigen to the osteoclast in transgenic mice causes osteoclast tumors and transformation and apoptosis of osteoclasts.
Osteoclasts are terminally differentiated cells that express tartrate-resistant acid phosphatase (TRAP) at a higher level than other normal cells. Therefore, in an attempt to develop immortalized osteoclasts, we produced two lines of transgenic mice in which expression of the simian virus 40 T antigen oncogene was targeted to osteoclasts using the TRAP gene promoter. Osteoclasts were increased in number in bones from both lines. More than 50% of them appeared morphologically transformed, 2-5% were mitotic, but, unexpectedly, 5% were apoptotic. Osteoclast tumors were observed occasionally in one line of mice (line 4), and sheets of TRAP-positive cells (tumorlets) developed in most mice in both lines. Although cells isolated from these tumorlets formed multinucleated TRAP-positive cells that resorbed bone in vitro, to date we have been unable to develop an immortalized osteoclast cell line from them. Osteoclasts from one line (line 5) had reduced ruffled border formation and a higher level of T-antigen expression than osteoclasts in the other line (line 4), and these features were associated with the presence of osteopetrosis. However, osteoclasts from these osteopetrotic mice and from line 4 mice resorbed bone normally when the mice were treated with interleukin-1. These findings indicate that T antigen can be targeted to osteoclasts in transgenic mice and causes osteoclast transformation, tumors, mitosis, and apoptosis. When T antigen is expressed at high levels, functional impairment of osteoclasts can be detected. Furthermore, these results suggest that T antigen is insufficient on its own to immortalize cells in the osteoclast lineage.
Acid Phosphatase↗
Effects of parathyroid hormone (PTH)-related protein and PTH on osteoclasts and osteoclast precursors in vivo.
Increased production of PTH-related protein (PTHrP) and PTH is frequently responsible for hypercalcemia and its associated morbidity. However, it is unclear whether these peptides produce identical effects on cells in the osteoclast lineage in vivo. To examine the effects of continuous in vivo exposure to these factors on both the osteoclast precursors and mature osteoclasts, we inoculated Chinese hamster ovarian cells expressing PTH-(1-84), PTHrP-(1-141), or nontransfected Chinese hamster ovarian cells into nude mice. The effects of these tumors on blood ionized calcium, plasma PTH and PTHrP concentrations, and osteoclast formation were then determined. PTH and PTHrP tumor-bearing mice became hypercalcemic (1.90 +/- 0.04 and 1.97 +/- 0.16 mmol/liter, respectively) compared with control mice (1.29 +/- 0.015 mmol/liter). After 4 days of hypercalcemia, mice were killed, and bone marrow cells were harvested to examine cells at three discrete stages of osteoclast development: multipotent osteoclast precursors, the granulocyte/macrophage colony-forming unit; more committed marrow mononuclear osteoclast precursors; and mature osteoclasts. Neither PTH nor PTHrP had an effect on granulocyte/macrophage colony-forming unit, but similarly increased the number of more committed mononuclear osteoclast progenitors as well as mature osteoclasts in the calvaria. No differences were detected between the effects of PTH and PTHrP on cells in the osteoclast lineage in vivo. Thus, PTH and PTHrP appear to affect only more differentiated cells in the osteoclast lineage, and the differences in osteoclastic bone resorption between primary hyperparathyroidism and humoral hypercalcemia of malignancy are probably due to mechanisms other than effects on osteoclast precursor cells in vivo.
Animals↗
An in vivo model of human multiple myeloma bone disease.
Osteolytic bone destruction and its complications, such as hypercalcemia, pathologic fractures and nerve compression, are the major source of morbidity in patients with multiple myeloma (MM). The bone destruction in MM is due to increased osteoclast activity, but the mechanisms responsible are not entirely clear. We have utilized a human plasma cell leukemia cell line, ARH-77, that has disseminated growth in mice with severe combined immunodeficiency (SCID) and expresses immunoglobulin G kappa (IgG kappa), as a model for human MM. Fifteen SCID mice were irradiated with 400R and 10 of these were injected with 10(6) ARH-77 cells i.v., 24 h after irradiation. Five mice were used as a control group. Development of bone disease was assessed by blood calcium levels, x-rays and histology. Seven out of seven mice that survived irradiation and received ARH-77 cells developed hind limb paralysis 28-35 days after injection. One hundred percent of these mice developed hypercalcemia (1.35-1.46 mmol/l), a mean of five days after becoming paraplegic. Lytic bone lesions were detected by x-ray in all the hypercalcemic mice examined. No lytic lesions or hypercalcemic developed in the controls. Mice were then sacrificed after developing hypercalcemia. Histologic examination of the ARH-77 mice showed infiltration of myeloma cells in the liver and spleen. Marked infiltration by the tumor was found in vertebrae and long bones, with loss of bony trabeculae and increased osteoclast numbers.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Mineralisation defects after pamidronate for Paget's disease.
Explore the source record for details and available documents.
Calcification, Physiologic↗