Search

A new series of bisphosphonic acids bearing nitrogen containing heterocycles and carbocycles has been designed and synthesized. The most potent novel bisphosphonates were shown to have lower minera...

// Theresa E. Hickey 1, * , Connie M. Irvine 1, * , Heidi Dvinge 2, 3 , Gerard A. Tarulli 1 , Adrienne R. Hanson 1 , Natalie K. Ryan 1 , Marie A. Pickering 1 , Stephen N. Birrell 1 , Dong Gui Hu 4 , Peter I. Mackenzie 4 , Roslin Russell 5 , Carlos Caldas 5 , Ganesh V. Raj 6 , Scott M. Dehm 7, 8 , Stephen R. Plymate 9 , Robert K. Bradley 2, 3 , Wayne D. Tilley 1, 10 , Luke A. Selth 1, 10 1 Dame Roma Mitchell Cancer Research Laboratories, Discipline of Medicine, The University of Adelaide, SA 5005, Australia 2 Computational Biology Program, Public Health Sciences Division, Seattle, WA 98109, USA 3 Basic Sciences Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA 4 Department of Clinical Pharmacology, Flinders University School of Medicine, Flinders Medical Centre, Bedford Park, SA 5042, Australia 5 Cancer Research UK Cambridge Research Institute, Li Ka Shing Centre, Robinson Way, Cambridge, CB2 0RE, UK 6 Department of Urology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA 7 Masonic Cancer Center, University of Minnesota, Minneapolis, MN 55905, USA 8 Department of Laboratory Medicine and Pathology, University of Minnesota, Minneapolis, MN 55905, USA 9 Department of Medicine and VAPSHCS, University of Washington, Seattle, WA 98109, USA 10 Freemasons Foundation Centre for Men’s Health, School of Medicine, The University of Adelaide, SA 5005, Australia * These authors have contributed equally to this work Correspondence to: Luke A. Selth, e-mail: luke.selth@adelaide.edu.au Wayne D. Tilley, e-mail: wayne.tilley@adelaide.edu.au Keywords: androgen receptor, breast cancer, androgen deprivation therapy, alternative splicing, biomarker Received: October 19, 2015 Accepted: October 26, 2015 Published: November 05, 2015 ABSTRACT The importance of androgen receptor (AR) signaling is increasingly being recognized in breast cancer, which has elicited clinical trials aimed at assessing the efficacy of androgen deprivation therapy (ADT) for metastatic disease. In prostate cancer, resistance to ADT is frequently associated with the emergence of androgen-independent splice variants of the AR (AR variants, AR-Vs) that lack the LBD and are constitutively active. Women with breast cancer may be prone to a similar phenomenon. Herein, we show that in addition to the prototypical transcript, the AR gene produces a diverse range of AR-V transcripts in primary breast tumors. The most frequently and highly expressed variant was AR-V7 (exons 1/2/3/CE3), which was detectable at the mRNA level in > 50% of all breast cancers and at the protein level in a subset of ERα-negative tumors. Functionally, AR-V7 is a constitutively active and ADT-resistant transcription factor that promotes growth and regulates a transcriptional program distinct from AR in ERα-negative breast cancer cells. Importantly, we provide ex vivo evidence that AR-V7 is upregulated by the AR antagonist enzalutamide in primary breast tumors. These findings have implications for treatment response in the ongoing clinical trials of ADT in breast cancer.

Summary Estrogen receptor α (ERα) is the key transcriptional driver in a large proportion of breast cancers. We report that APOBEC3B (A3B) is required for regulation of gene expression by ER and acts by causing C-to-U deamination at ER binding regions. We show that these C-to-U changes lead to the generation of DNA strand breaks through activation of base excision repair (BER) and to repair by non-homologous end-joining (NHEJ) pathways. We provide evidence that transient cytidine deamination by A3B aids chromatin modification and remodelling at the regulatory regions of ER target genes that promotes their expression. A3B expression is associated with poor patient survival in ER+ breast cancer, reinforcing the physiological significance of A3B for ER action., Graphical Abstract, Highlights • APOBEC3B is associated with poor survival in ER+ breast cancer patients • APOBEC3B controls breast cancer cell growth by promoting ER transcriptional activity • APOBEC3B can cause C-to-U mutations at ER target genes, to activate DNA repair • Repair of APOBEC3B-induced lesions allows chromatin remodelling that stimulates gene expression, Periyasamy et al. show that APOBEC3B is required for the regulation of gene expression by the estrogen receptor in breast cancer cells. They report APOBEC3B can promote cytidine deamination at gene regulatory regions, with consequent repair providing a mechanism for chromatin remodelling that facilitates gene expression.

Bisphosphonates are chemically stable analogs of inorganic pyrophosphate, which are resistant to breakdown by enzymatic hydrolysis. The biological effects of bisphosphonates on calcium metabolism were originally ascribed to their physico-chemical effects on hydroxyapatite crystals. Although such effects may contribute to their overall action, their effects on cells are probably of greater importance, particularly for the more potent compounds. Remarkable progress has been made in increasing the potency of bisphosphonates as inhibitors of bone resorption, and the most potent compounds in current use are characterized by the presence of a nitrogen atom at critical positions in the side chain which, together with the bisphosphonate moiety itself, seems to be essential for maximal activity. As a class the bisphosphonates offer a very effective means of treating Paget's disease.

Osteoblasts undergo apoptosis both in vitro and in vivo in response to high dose glucocorticoid (GC) treatment. However, the molecular mechanisms remain elusive, hindering the prevention and treatment of this side-effect. Apoptosis was induced by dexamethasone (Dex) in murine MBA-15.4 osteoblasts within 24-48 h of treatment. We found dose- and time-dependent upregulation of Bim protein, a pro-apoptotic Bcl-2 family member, with highest levels at 24-48 h for 1 microM Dex. This was also observed in primary human bone marrow stromal cells. Bim is subjected to stringent transcriptional and post-translational regulation in osteoblasts. Bim mRNA was upregulated in response to 1 microM Dex; both cycloheximide and the GC receptor antagonist, RU486, prevented Dex-induction of Bim protein, indicating transcriptional regulation involving the GC receptor. The proteasome inhibitor, MG132, potently increased Bim protein levels. Bim was also upregulated in osteoblasts undergoing apoptosis in response to serum deprivation and matrix detachment. Gene silencing experiments show that short interference RNA (siRNA) specific for Bim or the downstream effector Bax both reduced apoptosis induced by Dex in osteoblastic cells. These findings suggest that Bim is a novel regulator of osteoblast apoptosis and may be a therapeutic target.

1. The diphosphonate, disodium etidronate (disodium ethane-1-hydroxy-1,1-diphosphonate; EHDP), is known to increase plasma inorganic phosphate in man. The present study examines the mechanism of this effect. 2. When EHDP was given by mouth at a dose of 80 μmol (20 mg) kg−1 day−1, plasma phosphate was significantly increased 24 h after the first dose but did not reach its maximum value for 2–3 weeks. When the drug was stopped, plasma phosphate returned to pretreatment values within 3 weeks. 3. Urinary excretion rate of phosphate was not greatly changed during treatment with EHDP despite the large increase in plasma phosphate, suggesting an alteration in renal handling. This was examined directly by infusing phosphate and inulin in six patients off and on EHDP. 4. EHDP had no effect on glomerular filtration rate (GFR) but produced a large increase in the maximum rate of renal tubular reabsorption of phosphate (Tm,P). The ratio Tm,P/GFR increased from a mean value of 1.15 mmol/l to 2.10 mmol/l on EHDP. This increase accounted for the hyperphosphataemia. 5. The same amount of phosphate infused at the same rate produced a greater rise in plasma phosphate when patients were on EHDP than when they were not, indicating a reduced net rate of entry of phosphate into tissues other than kidney. 6. Fasting total plasma calcium concentration and urine calcium excretion rate were not significantly altered by EHDP but the ability of infused phosphate to decrease plasma calcium was diminished. 7. It is suggested that EHDP alters phosphate transport in kidney and other tissues by a mechanism which is probably independent of the known hormonal influences on phosphate metabolism.

Twelve patients with Paget's disease of bone were treated with high doses of disodium etidronate for one month and compared with patients given treatments for longer periods. The effects of treatment for one month with etidronate 20 mg/kg daily were indistinguishable from six months' continuous treatment with the same dose but significantly better than treatment with 5 mg/kg daily in suppressing biochemical indices of disease activity. Treatment for one month was associated with transient osteomalacia but sustained suppression of bone resorption. Short term treatments with high doses of disodium etidronate may maximise suppression of disease activity but decrease exposure to unwanted effects.

The long-term effects of vitamin D analogues and metabolites on renal function were assessed in 24 patients with and without chronic renal failure. Treatment for periods of 5-45 months did not adversely affect renal function in 10 of 11 patients with stable renal function, although transient hypercalcaemia did cause transient rises in plasma creatinine. Of 13 patients with progressive renal failure before treatment, vitamin D-like compounds or the vehicle used for their administration may have accelerated renal failure in 3 patients independently of changes in plasma calcium or phosphate. Particular difficulties in assessing the effects of vitamin D-like compounds in progressive renal disease are discussed.

The concentration of inorganic pyrophosphate (I.P.P.) has been determined in the plasma, urine, and synovial fluid of patients with pyrophosphate arthropathy (chondrocalcinosis articularis or pseudogout). The concentrations of I.P.P. in plasma and urine were normal in patients with pyrophosphate arthropathy. In contrast, the concentrations in synovial fluid were higher in patients with pyrophosphate arthropathy (0·41-3·76 μg. I.P.P. per ml.) than in patients with joint effusions due to other causes (0·06-0·32 μg. I.P.P. per ml.). The concentrations of calcium and alkaline phosphatase were lower in the synovial fluid of patients than of controls. The disturbance in I.P.P. metabolism in pyrophosphate arthropathy seems to be a local rather than generalised phenomenon.

In the space of a few weeks, four articles appeared in the The Lancet and JAMA suggesting that using 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase inhibitors (statins) is associated with increased bone mineral density (BMD) [1] and a reduced fracture risk [2-4]. The stimulus for these case-control studies came from reports that the statins have unexpected effects on bone, increasing bone formation in rodents [5]. These observations offered a new insight into the potential importance of the cholesterol synthesis pathway in bone turnover and future therapeutic opportunities.

Earlier studies have shown that inorganic pyrophosphate (PP i) inhibits the dissolution of hydroxyapatite crystals in vitro and it has been suggested that PP i might be a physiological regulator of bone resorption. In this study PP 1 and other phosphate compounds have been tested for their ability to inhibit bone resorption induced by parathyroid hormone in mouse calvaria and to inhibit the rise in plasma calcium induced by parathyroid hormone in thyroparathyroidectomised rats on a low calcium diet. Orthophosphate, pyrophosphate, polyphosphate and two polymeric phosphate inhibitors of phosphatases did not inhibit the resorption of calvaria or the rise in plasma calcium. In contrast, several phosphonates containing P-C-P bonds retarded the dissolution of hydroxyapatite crystals in vitro, and, at concentrations down to 1.6×10 -6M, inhibited bone resorption in tissue culture. Some diphosphonates also inhibited the rise in plasma calcium in thyroparathyroidectomised rats. One reason for the difference between the effects of compounds containing P-O-P and P-C-P bonds may be related to the greater resistance of the latter to chemical and enzymic hydrolysis. Phosphonates may provide a model for the effect of endogenous PP 1 in bone, and might be of use in elucidating provide a model for the effect of endogenous PP 1 in bone, and might be of use in elucidating mechanisms of bone formation and resorption and in the therapy of diseases that involve increased resorption of bone. © 1970 Springer-Verlag.

Bisphosphonates (BPs) inhibit bone resorption and are widely used for the treatment of bone diseases, including osteoporosis. BPs are also being studied for their effects on hydroxyapatite (HAP)-containing biomaterials. There is a growing appreciation that there are hitherto unexpected differences among BPs in their mineral binding affinities that affect their pharmacological and biological properties. To study these differences, we have developed a method based on fast performance liquid chromatography using columns of HAP to which BPs and other phosphate-containing compounds can adsorb and be eluted by using phosphate buffer gradients at pH 6.8. The individual compounds emerge as discrete and reproducible peaks for a range of compounds with different affinities. For example, the peak retention times (min; mean ± SEM) were 22.0 ± 0.3 for zoledronate, 16.16 ± 0.44 for risedronate, and 9.0 ± 0.28 for its phosphonocarboxylate analog, NE10790. These results suggest that there are substantial differences among BPs in their binding to HAP. These differences may be exploited in the development of biomaterials and may also partly explain the extent of their relative skeletal retention and persistence of biological effects observed in both animal and clinical studies. © 2009 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 2010

Bisphosphonates are now the most widely used drugs for diseases associated with increased bone resorption, such as osteoporosis. Although bisphosphonates act directly on osteoclasts, and interfere with specific biochemical processes such as protein prenylation, their ability to adsorb to bone mineral also contributes to their potency and duration of action. The aim of the present study was to compare the binding affinities for hydroxyapatite (HAP) of 6 bisphosphonates currently used clinically and to determine the effects of these bisphosphonates on other mineral surface properties including zeta potential and interfacial tension. Affinity constants (K(L)) for the adsorption of bisphosphonates were calculated from kinetic studies on HAP crystal growth using a constant composition method at 37 degrees C and at physiological ionic strength (0.15 M). Under conditions likely to simulate bisphosphonate binding onto bone, there were significant differences in K(L) among the bisphosphonates for HAP growth (pH 7.4) with a rank order of zoledronate > alendronate > ibandronate > risedronate > etidronate > clodronate. The measurements of zeta potential show that the crystal surface is modified by the adsorption of bisphosphonates in a manner best explained by molecular charges related to the protonation of their side-chain moieties, with risedronate showing substantial differences from alendronate, ibandronate, and zoledronate. The studies of the solid/liquid interfacial properties show additional differences among the bisphosphonates that may influence their mechanisms for binding and inhibiting crystal growth and dissolution. The observed differences in kinetic binding affinities, HAP zeta potentials, and interfacial tension are likely to contribute to the biological properties of the various bisphosphonates. In particular, these binding properties may contribute to differences in uptake and persistence in bone and the reversibility of effects. These properties, therefore, have potential clinical implications that may be important in understanding differences among potent bisphosphonates, such as the apparently more prolonged duration of action of alendronate and zoledronate compared with the more readily reversible effects of etidronate and risedronate.

The discovery and development of the bisphosphonates (BPs) as a major class of drugs for the treatment of bone diseases has been a fascinating journey that is still not over. In clinical medicine, several BPs are established as the treatments of choice for various diseases of excessive bone resorption, including Paget's disease of bone, myeloma and bone metastases, and osteoporosis. Bisphosphonates are chemically stable analogues of inorganic pyrophosphate, and are resistant to breakdown by enzymatic hydrolysis. Bisphosphonates inhibit bone resorption by being selectively taken up and adsorbed to mineral surfaces in bone, where they interfere with the action of the bone-resorbing osteoclasts. Bisphosphonates are internalized by osteoclasts and interfere with specific biochemical processes. Bisphosphonates can be classified into at least two groups with different molecular modes of action. The simpler non-nitrogen-containing bisphosphonates (such as clodronate and etidronate) can be metabolically incorporated into nonhydrolyzable analogues of adenosine triphosphate (ATP) that may inhibit ATP-dependent intracellular enzymes. The more potent, nitrogen-containing bisphosphonates (such as pamidronate, alendronate, risedronate, ibandronate, and zoledronate) are not metabolized in this way but can inhibit enzymes of the mevalonate pathway, thereby preventing the biosynthesis of isoprenoid compounds that are essential for the posttranslational modification of small GTP-binding proteins (which are also GTPases) such as rab, rho, and rac. The inhibition of protein prenylation and the disruption of the function of these key regulatory proteins explain the loss of osteoclast activity and induction of apoptosis. The key target for bisphosphonates is farnesyl pyrophosphate synthase (FPPS) within osteoclasts, and the recently elucidated crystal structure of this enzyme reveals how BPs bind to and inhibit at the active site via their critical N atoms. In conclusion, bisphosphonates are now established as an important class of drugs for the treatment of many bone diseases, and their mode of action is being unraveled. As a result their full therapeutic potential is gradually being realized.

The present study describes the biological effects of risedronate, a pyridinyl bisphosphonate, on bone and assesses the safety and tolerability of risedronate when given at high doses, with or without calcium, to postmenopausal women with spinal osteoporosis. This single-center descriptive, double-blind, placebo-controlled, randomized, parallel group study included 32 postmenopausal white women with at least one radiographically confirmed vertebral compression fracture. Patients were randomized to one of four different dose regimen groups: (i) R-P, risedronate 20 mg/day for 14 days, followed by placebo for 42 days; (ii) R-CP-P, risedronate 20 mg/day for 14 days, followed by elemental calcium 1000 mg/day and placebo for 14 days, then by placebo for 28 days; (iii) R-CP-R-CP, risedronate 20 mg/day for 7 days, followed by elemental calcium 1000 mg/day and placebo for 21 days, then risedronate 20 mg/day for 7 days, and finally elemental calcium 1000 mg/day and placebo for 21 days; and (iv) P, placebo for 56 days. The biological response was investigated by measuring serum calcium, parathyroid hormone (PTH), and 2 h urinary pyridinoline/creatinine (Pyr/Cr) and deoxypyridinoline/creatinine (DPyr/Cr) ratios at baseline and at days 3, 7, 14, 21, 28, 35, 42, 49, 56, and 84. Overall, there were no consistent trends observed between the active group and placebo for serum calcium. In groups R-P, R-CP-P, and R-CP-R-CP, mean serum PTH levels were elevated above baseline values for the entire 56 day treatment period and remained elevated, although to a lesser extent, at the day 84 follow-up visit. The effect of calcium supplementation on PTH was variable. Urinary Pyr/Cr and DPyr/Cr ratios were decreased from baseline over the entire study period in all groups receiving risedronate. The maximum observed percent decreases from baseline for Pyr/Cr and DPyr/Cr were -46.9% and -58.8%, respectively, at day 49 in the R-CP-R-CP group. In conclusion, risedronate given orally at a dose of 20 mg/day, continuously for 7 or 14 days, resulted in the expected biological response in osteoporotic women. The time course of changes in PTH levels following cessation of dosing was unaffected by calcium supplementation. There was no evidence of a PTH-mediated rebound in bone resorption following cessation of therapy. Furthermore, based on collagen cross-link data, patients did not show an excessive reduction in bone turnover.

Bovine articular chondrocytes were maintained in high density pellet cultures with and without serum and nucleotide triphosphates for different periods of time. Despite half-lives in culture of about 3 h, adenosine triphosphate and uridine triphosphate in the presence of serum increased sulphated glycosaminoglycan and collagen deposition above control levels. In the presence of serum a single dose of uridine triphosphate on the first day of culture was sufficient to induce significant increases in subsequent proteoglycan and collagen deposition. We conclude that both adenine triphosphate and uridine triphosphate are anabolic for articular chondrocytes, and that this effect on the chondrocyte is long-term.

BPs can be grouped into two general classes according to their chemical structure and the molecular mechanism by which they inhibit osteoclast-mediated bone resorption. The simple BPs can be metabolically incorporated into non-hydrolysable analogues of ATP that accumulate intracellularly in osteoclasts, causing osteoclast cell death by apoptosis. By contrast, the more potent N-BPs inhibit FPP synthase, an enzyme in the mevalonate pathway. Inhibition of this enzyme in osteoclasts prevents the biosynthesis of isoprenoid lipids that are required for the prenylation of small GTPase signalling proteins necessary for osteoclast function. Inhibition of FPP synthase in cells other than osteoclasts also appears to account for the adverse effects of N-BPs in vivo (including the acute phase reaction) and for the anti-tumour effects of N-BPs in vitro.

During reproduction, female teleost fish display increased plasma estrogen and greatly increased total plasma calcium concentrations; the main source of this calcium seems to be the scale. Osteonectin, a collagen-binding glycoprotein, is a major noncollagenous constituent of mammalian bone and is a product mainly of the osteoblasts. RT-PCT has been applied to clone and sequence part of the osteonectin gene from the goldfish, Carassius auratus. The use of a goldfish scale cell line (GFS) and a specific probe to goldfish osteonectin mRNA has allowed the study of the potential effects of estrogen and other calcitropic hormones on the cells derived from the scales. Osteonectin mRNA was detected in teleost bone, scale, and GFS cells by Northern blot analysis, hybridising to a transcript of approximately 1.6 kb. Expression of osteonectin mRNA was markedly down-regulated by 17beta-estradiol (10(-8) to 10(-11) M) in a dose-dependent fashion but was unaffected by calcitriol, TGFbeta, IL-1beta, calcitonin, and PTHrP. Down-regulation of osteonectin by estrogen is further evidence that estrogen participates in calcium homeostasis during vitellogenesis, acting directly on the cells responsible for matrix and mineral fluxes in scales.

Nitrogen-containing bisphosphonates were shown to cause macrophage apoptosis by inhibiting enzymes in the biosynthetic pathway leading from mevalonate to cholesterol. This study suggests that, in osteoclasts, geranylgeranyl diphosphate, the substrate for prenylation of most GTP binding proteins, is likely to be the crucial intermediate affected by these bisphosphonates. We report that murine osteoclast formation in culture is inhibited by both lovastatin, an inhibitor of hydroxymethylglutaryl CoA reductase, and alendronate. Lovastatin effects are blocked fully by mevalonate and less effectively by geranylgeraniol whereas alendronate effects are blocked partially by mevalonate and more effectively by geranylgeraniol. Alendronate inhibition of bone resorption in mouse calvaria also is blocked by mevalonate whereas clodronate inhibition is not. Furthermore, rabbit osteoclast formation and activity also are inhibited by lovastatin and alendronate. The lovastatin effects are prevented by mevalonate or geranylgeraniol, and alendronate effects are prevented by geranylgeraniol. Farnesol and squalene are without effect. Signaling studies show that lovastatin and alendronate activate in purified osteoclasts a 34-kDa kinase. Lovastatin-mediated activation is blocked by mevalonate and geranylgeraniol whereas alendronate activation is blocked by geranylgeraniol. Together, these findings support the hypothesis that alendronate, acting directly on osteoclasts, inhibits a rate-limiting step in the cholesterol biosynthesis pathway, essential for osteoclast function. This inhibition is prevented by exogenous geranylgeraniol, probably required for prenylation of GTP binding proteins that control cytoskeletal reorganization, vesicular fusion, and apoptosis, processes involved in osteoclast activation and survival.

Interleukin-6 (IL-6) is the major growth factor for human myeloma cells, exerting its effect through the IL-6 receptor (IL-6R). A soluble form of IL-6R (sIL-6R) has been identified, which increases the sensitivity of myeloma cells to IL-6. In patients with multiple myeloma (MM), serum concentrations of sIL-6R are elevated and associated with poor prognosis. The present study was undertaken to determine whether proteolytic cleavage of IL-6R could contribute to sIL-6R release from human myeloma cells, and also to identify the class of proteinase responsible for this event. Human myeloma cell lines were shown to express IL-6R upon their surface and also to release sIL-6R into culture supernatants. In addition, phorbol 12-myristate 13-acetate (PMA) stimulated a loss of IL-6R from the cell surface, with a corresponding increase in the concentration of sIL-6R in the supernatant. Inhibitors of serine and cysteine proteinases, and tissue inhibitor of metalloproteinase (TIMP) -1 and TIMP-2, were shown to have no effect on the magnitude of sIL-6R release. In contrast, TIMP-3 and a hydroxamate-based metalloproteinase inhibitor (BB-94), inhibited both constitutive and PMA-induced release of sIL-6R. Myeloma cells freshly isolated from the bone marrow of a patient with MM were also shown to express IL-6R upon their surface, and to shed this receptor in response to PMA. These data demonstrate that increased proteolytic cleavage of IL-6R, mediated by a non-matrix-type metalloproteinase, is likely to contribute to the elevated concentrations of sIL-6R found in the serum of patients with MM. Inhibition of sIL-6R release by hydroxamate-based metalloproteinase inhibitors may represent a novel therapeutic approach to the treatment of MM.

The etiology of osteoporosis is multifactorial, but there is evidence from both animal and human studies that the volume of marrow adipose tissue increases when bone volume is reduced in osteoporosis. The cell-related mechanism that may account for this inverse relationship between the volume of marrow adipose tissue and bone remains to be clarified, although it is known that both adipocytes and osteoblasts are derived from stromal cell precursors in bone marrow. We report that retroviral transduction with a temperature-sensitive oncogene (SV40 large T antigen) can generate bipotential cell lines from human marrow stroma that are capable of directed differentiation, in vitro, down either an osteogenic or adipocytic lineage pathway. One such clone, designated hOP 7, expresses type α1(I) procollagen and has low alkaline phosphatase (AP) activity under basal culture conditions that is reminiscent of an osteoprogenitor cell. Exposure of hOP 7 cells to dexamethasone upregulates AP activity and enables the cells to mineralize their extracellular matrix. Also, treatment with calcitriol induces osteocalcin expression and both PTH and PGE 2 induce/augment cAMP formation. Incubation with normal rabbit serum, however, causes the cells to become adipogenic as demonstrated by histological staining with Oil-red-O, expression of mRNA for the early and late adipocyte markers lipoprotein lipase and glycerol 3-phosphate dehydrogenase, respectively, and loss of type α1(I) procollagen mRNA. The generation of homogeneous populations of these cells, as confirmed by Southern blot analysis, demonstrates the capacity of a human clonal cell line to differentiate in either an osteogenic or adipogenic direction.

Purpose. The aim of the study was to determine whether bisphosphonates are internalised by Dictyosteliumamoebae and whether cellular uptake is required for their growth-inhibitory effects. Bisphosphonates inhibit growth of amoebae of the slime mould Dictyostelium discoideum, by mechanisms that appear to be similar to those that cause inhibition of osteoclastic bone resorption. Methods. Cell-free extracts prepared from amoebae that had been incubated with bisphosphonates were analysed by 3lP-n.m.r. spectroscopy or ion-exchange f.p.l.c., to identify the presence of bisphosphonates or bisphosphonate metabolites respectively. The growth-inhibitory effect of bisphosphonates towards Dictyostelium amoebae was also examined under conditions in which pinocytosis was inhibited. Results. All of the bisphosphonates studied were internalised by Dictyostelium amoebae, probably by fluid-phase pinocytosis, and could be detected in cell-free extracts. Amoebae that were prevented from internalising bisphosphonates by pinocytosis were markedly resistant to the growth-inhibitory effects of these compounds. In addition, bisphosphonates encapsulated within liposomes were more potent growth inhibitors of Dictyostelium owing to enhanced intracellular delivery of bisphosphonates. Conclusions. All bisphosphonates inhibit Dictyostelium growth by intracellular mechanisms following internalisation of bisphosphonates by fluid-phase pinocytosis. It is therefore likely that bisphosphonates also affect osteoclasts by interacting with intracellular, rather than extracellular, processes.

Short arm of chromosome 8 is a hot spot for chromosomal breaks, losses and amplifications in breast cancer. Although such genetic changes may have phenotypic consequences, the identity of candidate gene(s) remains to be clearly defined. Pol β gene is localized to chromosome 8p12‐p11 and encodes a key DNA base excision repair protein. Pol β may be a tumour suppressor and involved in breast cancer pathogenesis. We conducted the first and the largest study to comprehensively evaluate pol β in breast cancer. We investigated pol β gene copy number changes in two cohorts (n = 128 & n = 1952), pol β mRNA expression in two cohorts (n = 249 & n = 1952) and pol β protein expression in two cohorts (n = 1406 & n = 252). Artificial neural network analysis for pol β interacting genes was performed in 249 tumours. For mechanistic insights, pol β gene copy number changes, mRNA and protein levels were investigated together in 128 tumours and validated in 1952 tumours. Low pol β mRNA expression as well as low pol β protein expression was associated high grade, lymph node positivity, pleomorphism, triple negative, basal‐like phenotypes and poor survival (ps

The aim of this study was to investigate the effects of insulin-like growth factor-I, transforming growth factor-beta (TGF-beta), and interluekin-1 alpha (IL-1 alpha) on the deposition and degradation of a cartilage-like matrix in high-density pellet cultures of adult bovine chondrocytes. Proteoglycan was determined by toluidine blue staining and colorimetric assay. Type II collagen was determined by immunohistochemical staining and its unwinding in situ by a recently developed immunoassay. Bovine nasal chondrocytes cultured as pellets deposited a well-organized extracellular matrix of proteoglycan and type II collagen. Insulin-like growth factor-I (2-10 ng/ml) increased the synthesis and incorporation into the matrix of both these proteins. TGF-beta (2-10 ng/ml) also increased proteoglycan synthesis. However it inhibited proteoglycan deposition, presumably through increased degradation of the molecule, as shown by increased release of aggrecan fragments into the tissue culture medium. TGF-beta had no effect on type II collagen deposition. In pellet cultures of bovine nasal or articular chondrocytes, 20 ng/ml IL-1 alpha induced a significant degradation of both proteoglycan and type II collagen. The effect on collagen clearly involved proteolytic cleavage of its triple helix because there was an increase in the proportion of unwound type II collagen in the matrix, as well as a loss of total type II collagen. In explant cultures of intact bovine articular cartilage, incubation with 50 ng/ml IL-1 alpha stimulated significant degradation of the proteoglycan but no degradation of the type II collagen. These results demonstrate that although the articular chondrocytes are capable of degrading type II collagen when isolated, they do not do so in situ, presumably because of some inherent property of the mature extracellular matrix. This study demonstrates the utility of pellet cultures when investigating chondrocyte-mediated turnover of cartilage matrix and its modulation by cytokines and growth factors.

Bisphosphonates are synthetic pyrophosphate analogues and are therapeutic inhibitors of bone resorption, although their exact mechanisms of action are unclear. Some bisphosphonates can be metabolised into non-hydrolysable ATP analogues by Dictyostelium discoideum amoebae, in a back-reaction catalysed by several Class II aminoacyl-tRNA synthetases. We have found that the same enzymes in cell-free extracts of several human cell lines are also capable of metabolising in vitro the same bisphosphonates that are metabolised by Dictyostelium. These results indicate that human cells, following drug internalisation, should be capable of metabolising certain bisphosphonates. The toxic effects of these bisphosphonates towards bone-resorbing osteoclasts may therefore be due to accumulation of non-hydrolysable ATP analogues or inhibition of aminoacyl-tRNA synthetase enzymes.

Cathepsin B activity and its regulation by interleukin 1 beta (IL-1 beta) and parathyroid hormone (PTH) was investigated in normal human osteoblast-like cells (hOB) and in the human osteoblastic osteosarcoma cell line MG-63. Cathepsin B activity was measured using a fluorescent synthetic substrate, 7-N-benzyloxycarbonyl-L-arginyl-L-arginylamide-4-methylcoumarin, and its specificity was checked with E-64, a specific inhibitor of cysteine proteinases and CA074, a specific inhibitor of the enzyme. Cathepsin B activity was detected in crude extracts of cell monolayers and in conditioned media. In both cell types, basal activity was detected essentially in cell extracts, since in media only approximately 1.2% (hOB) and approximately 6% (MG-63) of the total activity was released. IL-1 beta (1-100 U/ml) and PTH (10(-9) M-10(-6) M) significantly stimulated cathepsin B activity in cell extracts and in conditioned media. In both cell types, the increase in proteolytic activity appeared to require RNA and protein synthesis after adding IL-1 beta or PTH. Using the above substrate, we also evaluated some biochemical properties of the enzyme, and its pH-stability and pH-optimum. In both cell types, intracellular cathepsin B activity was not resistant to neutral or slightly alkaline pH, whereas extracellular cathepsin B activity was stable. This study provides evidence that osteoblast-like cells produce and secrete active cathepsin B. The production and secretion was stimulated by IL-1 beta and PTH. The physiological role of cathepsin B produced by osteoblasts and stimulated by the bone resorbing agents remains to be elucidated. Since extracellular activity is stable under relatively physiological conditions, it is possible that the extracellular as well as intracellular form of the enzyme may play a role in matrix turnover.

In this study we tested the hypothesis that exercise induces an adaptive response in the developing skeleton which may be monitored in vivo by measuring biochemical markers of bone metabolism. The effects of exercise on two biochemical markers of bone formation were determined; the carboxyterminal propeptide of type I procollagen (PICP), and the bone-specific isoenzyme of alkaline phosphatase (BAP), and one putative marker of resorption, the pyridinoline crosslinked telopeptide domain of type I collagen (ICTP). All three markers were measured for a year in 2-year-old thoroughbred horses exercised three times a week on a treadmill, and values compared to a control group of age-matched animals. Levels of all three markers fell in both exercised and control groups over the 12-month period reflecting normal age changes. However, there were differences between groups in the pattern of this decrease. When expressed as a percentage of baseline values, BAP was higher (p < 0.05) at 2 months and both BAP and the PICP were higher at 4 months (p < 0.01 and p < 0.05, respectively) in the exercised group, reflecting an increase in bone turnover in this group in the early stages of training. PICP levels were also elevated in the exercised group at 10 months and this result indicates an increase in bone turnover at this time. The changes in ICTP were different; at 2 months, levels were higher in exercised animals than in controls, but there was no significant difference between the two groups at 4 and 6 months. After 8 months, ICTP levels in the exercised group increased returning to near baseline values at 10 months. In summary, the results of these assays for bone ALP and metabolites of type I collagen indicate that the treadmill exercise regimen used for this study resulted in a general increase in bone turnover in 2-year-old thoroughbreds. These findings indicate that biochemical marker determinations may provide a sensitive, noninvasive method of monitoring skeletal turnover during athletic training.

Biochemical markers of bone metabolism were analysed in serum samples obtained from 60 horses with no history of orthopaedic disease (age 3 months-20 years). Serum levels of the carboxyterminal propeptide of type I procollagen (PICP), a marker of bone formation and the pyridinoline cross linked telopeptide domain of type I collagen (ICTP), a putative marker of bone resorption, were measured by radioimmunoassay (RIA). Serum levels of the bone specific isoenzyme of alkaline phosphatase (BALP), another marker of bone formation, were measured by a wheatgerm agglutinin affinity (WGA) method. Total alkaline phosphatase levels were also determined. Serum levels of PICP were significantly correlated with bone ALP (r = 0.78, P < 0.0001) and ICTP (r = 0.87, P < 0.0001). ICTP levels also correlated significantly with bone ALP (r = 0.81, P < 0.0001). However, total alkaline phosphatase did not correlate significantly with PICP, ICTP and BALP in horses over 1 year of age. There was an inverse correlation between serum levels of all biochemical markers and age of animals, with the most significant changes seen over the first 2 years. In animals less than 1 year of age, the reference ranges (mean +/- s.d. 1.96) were as follows: PICP 1216-2666 micrograms/l, ICTP 13.8-26.7 micrograms/l, bone ALP 134-288 u/l and total ALP 223-498 u/l. In 2-year-olds, the equivalent reference ranges were: PICP 550-1472 micrograms/l, ICTP 7.96-22.8 micrograms/l, bone ALP 32.7-125 u/l and total ALP 134-238 u/l.(ABSTRACT TRUNCATED AT 250 WORDS)

Type II collagen and aggrecan are major components of the extracellular matrix of articular cartilage. Their biosynthesis and catabolism are regulated by chondrocytes. They may be used as markers of chondrocyte phenotype for cells cultured in vitro. Type II collagen gene expression was detected by amplification of type II collagen-specific sequences, using cDNA produced by reverse transcription of mRNA extracted from freshly isolated and cultured human articular chondrocytes by the polymerase chain reaction (PCR). The synthesis of gene product was confirmed by immunohistochemical localization of type II collagen in cartilage sections and in cultured chondrocytes. Aggrecan core protein was also immunolocalized in cartilage sections and in chondrocytes in culture. Expression of type II collagen or aggrecan was not detected immunohistochemically in skin or bone. These results demonstrate that human articular chondrocytes can be characterized in culture, by the combined application of PCR and immunohistochemistry. Interleukin-1beta (IL-1beta) may play an important role in the destruction of cartilage matrix in arthritis, whereas transforming growth factor-beta (TGFbeta) may have an opposing effect and their combined actions may modulate chondrocyte phenotype. The effect of rhIL-1beta and rhTGFbeta on the production of type II collagen by chondrocytes in culture was investigated. It was shown that TGFbeta enhanced the production of type II collagen, localized immunocytochemically, in cultured chondrocytes. IL-1beta inhibited expression of mRNA for type II collagen. The implications of this study, in terms of a better understanding of degenerative cartilage disease, are discussed.

The aims of the study were to evaluate the use of bone-specific biochemical markers of turnover in type I osteoporosis, to test for evidence of heterogeneity of bone turnover in this condition, and to attempt to devise an ‘uncoupling index’ by using the relationship between bone-specific biochemical markers of bone formation and bone resorption. In women with type I osteoporosis (mean age 64 years, SD 5;n=63) the mean level of serum osteocalcin, a specific biochemical marker of bone formation, was 9.9 ng/ml (SD 2.0), which was higher than the level in normal postmenopausal women (mean age 65 years, SD 6;n=8.9 ng/ml (SD 2.0;p

The 24 h urinary excretion of the collagen degradation products pyridinoline (Pyr) and deoxypyridinoline (Dpyr) have been proposed as specific and quantitative indices of bone resorption. We compared the value of the urinary excretion of Pyr and Dpyr to that of hydroxyproline (OHP) in 11 patients with Paget's disease of bone before and during treatment with inhibitors of bone resorption, during admission to a metabolic ward and maintenance on a gelatin-free diet. Pyr and Dpyr excretion rates were significantly correlated with those of OHP (r = 0.81 and 0.77, respectively, P0.001; n = 106). The rate and degree of suppression of bone resorption were monitored in 6 of the patients similarly treated with intravenous dimethyl-APD at a dose of 4 mg/day for 10 days, by daily measurements of the 24 h urinary excretion of Pyr, Dpyr and OHP. Treatment with dimethyl-APD resulted in a decrease in the three indices of bone resorption. The percentage change from baseline values was similar for the three indices, although changes in Dpyr appeared to follow more closely those of OHP. Our findings suggest that Pyr and Dpyr are useful and specific indices of bone resorption in Paget's disease of bone. They appear to confer no advantage, however, over the traditional determination of the urinary excretion of OHP, in the monitoring of response to treatment of such patients with inhibitors of bone resorption. Further studies are required to establish the value of these new biochemical indices of bone resorption, possibly in more subtle disorders of bone metabolism such as osteoporosis.

The measurement of the 3–OH pyridinium compounds, pyridinoline (Pyr) and deoxypyridinoline (Dpyr), in urine by high performance liquid chromato-graphy is potentially useful in clinical studies, since they are specific biochemical markers of bone resorption. The aims of the present study were to improve assay performance and optimize sample collection. An isocratic high performance liquid chromatogram (HPLC) separation with baseline resolution was accomplished within 4 min using heptafluorobutyric acid as an ion-pair. The sample preparation for HPLC, using CFl cellulose, produced uncontami-nated samples with a recovery higher than 90% for both crosslinks. An elastin-derived material, tentatively identified as isodesmosine (Ides), was also tested and proved to be a suitable internal standard. Use of this standard improved assay precision. The effect of an oral gelatin load on the excretion of Pyr and Dyr was investigated. The creatinine corrected excretion of Pyr and Dpyr was unchanged over a 6 h period, in contrast to the 10–fold increase in the excretion of urinary hydroxyproline with a peak 2–4 h after ingestion. In 20 postmenopausal women, 2 h fasting morning urine results correlated with results from 24–h urine collections Dpyr/Cr (r = 0.70, n= 20). There was a day-to-day variation of 26% in adults studied for 10 days.

Retinoic acid receptor-alpha (RAR alpha) is a known estrogen target gene in breast cancer cells. The consequence of RAR alpha induction by estrogen was previously unknown. We now show that RAR alpha is required for efficient estrogen receptor-alpha (ER)-mediated transcription and cell proliferation. RAR alpha can interact with ER-binding sites, but this occurs in an ER-dependent manner, providing a novel role for RAR alpha that is independent of its classic role. We show, on a genome-wide scale, that RAR alpha and ER can co-occupy regulatory regions together within the chromatin. This transcriptionally active co-occupancy and dependency occurs when exposed to the predominant breast cancer hormone, estrogen--an interaction that is promoted by the estrogen-ER induction of RAR alpha. These findings implicate RAR alpha as an essential component of the ER complex, potentially by maintaining ER-cofactor interactions, and suggest that different nuclear receptors can cooperate for effective transcriptional activity in breast cancer cells.

The utility of measurement of the urinary excretion of the 3-hydroxypyridinium cross-links, pyridinoline (Pyr) and deoxypyridinoline (Dpyr) as indices of bone resorption in rats was investigated. Total Pyr and Dpyr excretion were measured in young rats treated by s.c. infusion with rat parathyroid hormone (1-34) (PTH) at 22-30 micrograms/kg/day or with diluent (controls) for 14 days. During infusion, average urinary excretion of both cross-links was significantly higher in PTH rats (Pyr: 11.77 +/- 0.44 nmol/day), Dpyr: 15.81 +/- 0.95 nmol/day) than in controls (Pyr: 10.17 +/- 0.35 nmol/day, Dpyr: 12.03 +/- 0.67 nmol/day). These results were consistent with the magnitude of the expected increase in bone resorption rate with this dose of PTH. The method appears to provide a sensitive measure of bone resorption for in vivo bone studies in rats.

Hypophosphatasia is a rare inherited disorder in which the activity of the bone/liver/kidney or tissue nonspecific form of alkaline phosphatase (ALP) is reduced. The clinical expression of the disease is highly variable, but in early life the severity tends to reflect the age of onset. Accordingly, the disease is often classified into perinatal, infantile, and childhood forms. Hypophosphatasia also occurs in adults. Some exhibit symptoms in adulthood for the first time, but others have a history of the disease in early life with an intervening symptom-free period. Defective mineralization of bones and teeth is the predominant clinical feature of all forms of the disease. Biochemically, the reduction in ALP activity is associated with alterations in the extracellular metabolism of various phosphorylated compounds, including inorganic pyrophosphate (PPi), phosphoethanolamine, and pyridoxal 5'-phosphate. Of these, PPi may have an especially important role in the development of the mineralization defect. Accordingly, the extracellular metabolism of PPi and its possible role in the regulation of mineralization will be discussed.

It has been shown in several laboratories that addition of beta-glycerophosphate (beta GP), a substrate for alkaline phosphatase (AP), to cultured osteoblast-like cells induces deposition of orthophosphate (Pi) and Ca within seven days. Even though this effect is regarded as an in vitro model of bone mineralization, it is not known whether it is specific for osteoblasts. We have, therefore, studied the amounts of Pi and Ca deposited after seven days with 10 mM beta GP in culture wells containing confluent cultures of osteoblast-like cells (OB) derived from human trabecular bone explants, human skin fibroblasts (SF), or culture medium alone (MED). Ox liver AP at an activity considerably greater than the endogenous AP activity of the cells, but comparable with that of other osteoblast models, was added to ensure a similar rate of Pi generation from beta GP in all wells. beta GP was converted quantitatively to Pi within seven days, leading to a nonphysiological 10-fold increase in the Pi concentration in the culture medium. After thorough rinsing on day seven, the OB and SF wells contained deposits of Pi and Ca, but the amounts were comparable for the two cell types. Smaller, but significant, amounts of Pi and Ca were also detectable even in rinsed MED wells. This suggests that the detection of such deposits in beta GP experiments cannot necessarily be interpreted as a specific property of osteoblast cultures in vitro, and may simply reflect the presence of AP.(ABSTRACT TRUNCATED AT 250 WORDS)

Apomine, a 1,1-bisphosphonate-ester with antitumor activity, has previously been reported to strongly down-regulate 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase), the rate-limiting enzyme in the mevalonate pathway responsible for the prenylation of proteins. Here, we show that although apomine down-regulated HMG-CoA reductase protein levels in myeloma cells, it did not inhibit protein prenylation, and apomine-induced apoptosis could not be prevented by mevalonate, indicating that apomine cytotoxicity is independent from its effects on HMG-CoA reductase. Instead, apomine cytotoxicity was prevented by the addition of phosphatidylcholine, which is similar to the previously reported ability of phosphatidylcholine to overcome the cytotoxicity of farnesol, whereas phosphatidylcholine had no effect on down-regulation of HMG-CoA reductase by apomine. These findings raised the possibility that apomine, independent from its own cytotoxic effects, could enhance the antitumor effects of the competitive HMG-CoA reductase inhibitor lovastatin via down-regulating HMG-CoA reductase. Indeed, treatment with apomine in combination with lovastatin resulted in synergistic decreases in viable cell number and induction of apoptosis. At the concentrations used, apomine down-regulated HMG-CoA reductase protein levels without being cytotoxic. Accumulation of unprenylated Rap1A by lovastatin was enhanced in the presence of apomine. Furthermore, synergy was completely prevented by mevalonate, and apomine did not synergize with desoxolovastatin, which does not inhibit HMG-CoA reductase. We conclude that the synergistic drug interaction results from an enhancement by apomine of the effects of lovastatin, mediated by down-regulation of HMG-CoA reductase by apomine. Thus, these findings demonstrate a novel strategy for enhancing the antitumor effects of lovastatin.

The bisphosphonates (BPs) are well established as the treatments of choice for disorders of excessive bone resorption, including Paget's disease of bone, myeloma and bone metastases, and osteoporosis. There is considerable new knowledge about how BPs work. Their classical pharmacological effects appear to result from two key properties: their affinity for bone mineral and their inhibitory effects on osteoclasts. Mineral binding affinities differ among the clinically used BPs and may influence their differential distribution within bone, their biological potency, and their duration of action. The inhibitory effects of the nitrogen-containing BPs (including alendronate, risedronate, ibandronate, and zoledronate) on osteoclasts appear to result from their inhibition of farnesyl pyrophosphate synthase (FPPS), a key branch-point enzyme in the mevalonate pathway. FPPS generates isoprenoid lipids used for the posttranslational modification of small GTP-binding proteins essential for osteoclast function. Effects on other cellular pathways, such as preventing apoptosis in osteocytes, are emerging as other potentially important mechanisms of action. As a class, BPs share several common properties. However, as with other classes of drugs, there are obvious chemical, biochemical, and pharmacological differences among the various individual BPs. Each BP has a unique profile that may help to explain potential important clinical differences among the BPs, in terms of speed of onset of fracture reduction, antifracture efficacy at different skeletal sites, and the degree and duration of suppression of bone turnover. As we approach the 40th anniversary of the discovery of their biological effects, there remain further opportunities for using their properties for medical purposes.

Osteoporosis and low bone mass are currently estimated to be a major public health risk affecting >50% of the female population over the age of 50. Because of their bone-selective pharmacokinetics, nitrogen-containing bisphosphonates (N-BPs), currently used as clinical inhibitors of bone-resorption diseases, target osteoclast farnesyl pyrophosphate synthase (FPPS) and inhibit protein prenylation. FPPS, a key branchpoint of the mevalonate pathway, catalyzes the successive condensation of isopentenyl pyrophosphate with dimethylallyl pyrophosphate and geranyl pyrophosphate. To understand the molecular events involved in inhibition of FPPS by N-BPs, we used protein crystallography, enzyme kinetics, and isothermal titration calorimetry. We report here high-resolution x-ray structures of the human enzyme in complexes with risedronate and zoledronate, two of the leading N-BPs in clinical use. These agents bind to the dimethylallyl/geranyl pyrophosphate ligand pocket and induce a conformational change. The interactions of the N-BP cyclic nitrogen with Thr-201 and Lys-200 suggest that these inhibitors achieve potency by positioning their nitrogen in the proposed carbocation-binding site. Kinetic analyses reveal that inhibition is competitive with geranyl pyrophosphate and is of a slow, tight binding character, indicating that isomerization of an initial enzyme–inhibitor complex occurs with inhibitor binding. Isothermal titration calorimetry indicates that binding of N-BPs to the apoenzyme is entropy-driven, presumably through desolvation entropy effects. These experiments reveal the molecular binding characteristics of an important pharmacological target and provide a route for further optimization of these important drugs.

Cathepsin B is a lysosomal cysteine proteinase, thought to be involved in the degradation of connective tissue breakdown products internalized by endocytosis. It has also been implicated in the extracellular matrix degradation of collagens and proteoglycans in disease states such as tumour invasion, rheumatoid arthritis and osteoarthritis. To date it is still unclear which factors can potentially modulate the release and/or activation of this enzyme.

Familial autosomal dominant calcium pyrophosphate dihydrate (CPPD) chondrocalcinosis has previously been mapped to chromosome 5p15. We have identified a mutation in the ANKH gene that segregates with the disease in a family with this condition. ANKH encodes a putative transmembrane inorganic pyrophosphate (PPi) transport channel. We postulate that loss of function of ANKH causes elevated extracellular PPi levels, predisposing to CPPD crystal deposition.

The processes of bone formation and resorption can be monitored in vivo by measuring enzymes and other protein products released by osteoblasts and osteoclasts respectively. The major validated biochemical markers of bone formation currently in use include the bone isoenzyme of alkaline phosphatase, osteocalcin (also known as BGP, bone Gla protein) and propeptides derived from the N or C terminal ends of the Type I procollagen molecule. The most useful markers of bone resorption are breakdown products of Type I collagen. The longest established of these is the measurement in urine of hydroxyproline in collagen peptides, but the assays are cumbersome. Furthermore, hydroxyproline is not specific to bone collagen and is also derived from the diet. There is therefore much current interest in collagen products that are more specific to bone, including galactosyl hydroxylysine, and the collagen crosslinks, pyridinoline and deoxypyridinoline. The pyridinolines and peptides derived from crosslinked regions in collagens appear to be the most promising markers of resorption and enable quantitative evaluation of rates of bone resorption in man. These biochemical methods are of use in the diagnosis and evaluation of bone diseases, in population studies, and for monitoring responses to hormones and drugs in clinical studies. It is important to remember that individual markers reflect different biochemical and physiological processes and may not, therefore, always show identical changes. There is an increasing amount of work being devoted to the study of bone biomarkers in osteoporosis. In population studies biochemical measurements may predict rates of bone loss and occurrence of fractures. However their value in the diagnosis and management of individual patients is less clear, partly because of the considerable biological and analytical variation in their measurement. There are exciting challenges ahead for improvements in technical methods and for the use of new markers derived from bone cells and bone matrix.

In a recent genome-wide study to identify androgen receptor target genes we found that the AR promotes tumor growth by enhancing the expression and activity of enzymes required for metabolic biosynthesis. This raises the possibility for effective therapeutic approaches directed towards AR target genes. Kinases are well-established drug targets. In this study we identify androgen regulated kinome and show that an important androgen regulated metabolic kinase is a drug target in prostate cancer. Our proteome study shows that this kinase interacts with the androgen receptor and regulates AR stability. Inhibition of this kinase significantly reduces AR protein levels and suppresses prostate cancer growth in cell-lines and ex vivo cultures. In clinical prostate cancer this kinase is highly over-expressed in metastatic prostate cancers and inhibition represses metastatic potential of prostate cancer cells. Our study therefore establishes this kinase for the first time as an AR target gene that regualtes AR itself as part of feedback loop and makes a case for further investigation of its inhibitors as novel AR antagonists in both localised and advanced disease. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B133. Citation Format: Mohammad asim, Charlie Massie, Heather Zecchini, Ajoeb Baridi, Nelma Gomes, Hisham Mohammed, Vincent Zecchini, Basetti Madhu, Arham Qureshi, Roslin Russell, Wiebke Hessenkemper, Rouchelle Sriranjan, Carrie Yang, Andy Lynch, Elena Gregorengko, Rory Stark, Paul Rennie, John Griffiths, Aria Baniahmad, Jasol Carroll, Ian Mills, David Neal. Regulation of the androgen receptor function by a metabolic kinase in prostate cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B133.

Bisphosphonates (BPs) are an important class of antiresorptive drugs used in the treatment of bone diseases, including osteoporosis. Although their mechanism of action has not been identified at the molecular level, there is substantial evidence that BPs can have a direct effect on osteoclasts by mechanisms that may lead to osteoclast cell death by apoptosis. BPs can also inhibit proliferation and cause cell death in macrophages in vitro. We have now shown that the toxic effect of BPs on macrophages is also due to the induction of apoptotic, rather than necrotic, cell death. Morphological and biochemical features that are definitive of apoptosis (chromatin condensation, nuclear fragmentation, and endonuclease-mediated internucleosomal cleavage of DNA) could be identified in mouse macrophage-like J774 and RAW264 cells, following treatment with 100 microM pamidronate, alendronate, and ibandronate for 24 h or more. Clodronate was much less potent, even at 2000 microM, while 2000 microM etidronate did not cause apoptosis. Apoptosis was not due to increased synthesis of nitric oxide and could not be prevented by inhibitors of nitric oxide synthases. Since macrophages, like osteoclasts, are particularly susceptible to BPs, these observations support the recent suggestion that the mechanism by which BPs inhibit bone resorption may involve osteoclast apoptosis. Furthermore, the macrophage-like cell lines used in this study may be a convenient model with which to identify the molecular mechanisms by which BPs promote apoptosis in osteoclasts. Induction of macrophage apoptosis by BPs in vivo may also account, at least in part, for the anti-inflammatory properties of BPs as well as the ability of BPs to cause an acute phase response.

For many years total alkaline phosphatase (AP) activity in serum has been used to monitor bone metabolism in different species. However, total AP lacks bone specificity because the total activity in serum is made up of several isoenzymes, of which the liver and bone isoforms predominate. The aim of the present study was to evaluate an immunoradiometric assay for measuring bone specific alkaline phosphatase (BAP) in horses. BAP, a specific marker of bone formation, was measured in sera from thoroughbred horses by using a previously characterised wheat germ lectin (WGL) precipitation assay and an immunoradiometric assay. The levels of immunoreactive BAP (iBAP) and WGL precipitated BAP (wBAP) were related to the serum levels of total AP and another marker of bone formation, the carboxy-terminal propeptide of type 1 collagen (PICP). In horses over one year old, iBAP correlated at least as strongly with total AP as with wBAP, which suggests that the immunoradiometric assay may partially cross-react with liver alkaline phosphatase in horse serum. This possibility was supported by the observation that there was a weaker correlation between iBAP and PICP than between wBAP and PICP. These data indicate that WGL precipitation is currently the most specific method for measuring bone specific alkaline phosphatase in horses.

Bisphosphonates are a class of synthetic pyrophosphate analogues. Some are known to be potent inhibitors of osteoclast-mediated bone resorption in vivo, but their mechanisms of action are unclear. The order of potency of bisphosphonates as inhibitors of bone resorption closely matches the order of potency as inhibitors of growth of amoebae of the slime mould Dictyostelium discoideum, indicating that bisphosphonates may have a mechanism of action that is similar in both osteoclasts and Dictyostelium. Methylenebisphosphonate and several halogenated derivatives, which have low potency as antiresorptive agents and as growth inhibitors of Dictyostelium, are metabolized intracellularly by Dictyostelium amoebae into methylene-containing adenine nucleotides. We have used a combination of n.m.r. and f.p.l.c. analysis to determine whether incorporation into nucleotides is a feature of other bisphosphonates, especially those that are potent antiresorptive agents. Only bisphosphonates with short side chains or of low potency are incorporated into adenine nucleotides, whereas those with long side chains or of high potency are not metabolized. Bisphosphonate metabolism in cell-free extracts of Dictyostelium was accompanied by inhibition of aminoacylation of tRNA by several aminoacyl-tRNA synthetases. These enzymes were barely affected by the bisphosphonates that were not metabolized. The results indicate that some bisphosphonates are not metabolically inert analogues of pyrophosphate and appear to be metabolized by aminoacyl-tRNA synthetases. The cellular effects of some bisphosphonates may be the result of their incorporation into adenine nucleotides or inhibition of aminoacyl-tRNA synthetases, although the potent bisphosphonates appear to act by a different mechanism.