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Although alfacalcidol has been widely used for the treatment of osteoporosis in certain countries, its mechanism of action in bone, especially in the vitamin D-replete state, remains unclear. Here we provide histomorphometric as well as biochemical evidence that alfacalcidol suppresses osteoclastic bone resorption in an ovariectomized rat model of osteoporosis. Furthermore, when compared with 17beta-estradiol, a representative antiresorptive drug, it is evident that alfacalcidol causes a dose-dependent suppression of bone resorption, and yet maintains or even stimulates bone formation, as reflected in increases in serum osteocalcin levels and bone formation rate at both trabecular and cortical sites. 17beta-Estradiol, which suppresses bone resorption to the same extent as alfacalcidol, causes a parallel reduction in the biochemical and histomorphometric markers of bone formation. As a final outcome, treatment with alfacalcidol increases bone mineral density and improves mechanical strength more effectively than 17beta-estradiol, with a more pronounced difference in cortical bone. We conclude that estrogens depress bone turnover primarily by suppressing bone resorption and, as a consequence, bone formation as well, whereas alfacalcidol "supercouples" these processes, in that it suppresses bone resorption while maintaining or stimulating bone formation.

We investigated the roles of interleukin-6 (IL-6) and parathyroid hormone-related peptide (PTHrP) in oral squamous cell carcinoma (OSCC)-induced osteoclast formation. Microarray analyses performed on 43 human OSCC specimens revealed that many of the specimens overexpressed PTHrP mRNA, but a few overexpressed IL-6 mRNA. Immunohistochemical analysis revealed that IL-6 was expressed not only in cancer cells but also in fibroblasts and osteoclasts at the tumor-bone interface. Many of the IL-6-positive cells coexpressed vimentin. Conditioned medium (CM) derived from the culture of oral cancer cell lines (BHY, Ca9-22, HSC3, and HO1-u-1) stimulated Rankl expression in stromal cells and osteoclast formation. Antibodies against both human PTHrP and mouse IL-6 receptor suppressed Rankl in ST2 cells and osteoclast formation induced by CM from BHY and Ca9-22, although the inhibitory effects of IL6 antibody were greater than those of PTHrP antibody. CM derived from all of the OSCC cell lines effectively induced IL-6 expression in stromal cells, and the induction was partially blocked by anti-PTHrP antibody. Xenografts of HSC3 cells onto the periosteal region of the parietal bone in athymic mice presented histology and expression profiles of RANKL and IL-6 similar to those observed in bone-invasive human OSCC specimens. These results indicate that OSCC provides a suitable microenvironment for osteoclast formation not only by producing IL-6 and PTHrP but also by stimulating stromal cells to synthesize IL-6.

Proteoglycans synthesized by osteoblasts are incorporated into bone matrix and thought to play a role in bone metabolism. Transforming growth factor (TGF) beta affects the synthesis of matrix proteins, including proteoglycans, in various stromal cells, and proteoglycans, especially decorin, are associated with matrix collagen. In the present study, the effects of TGF-beta 1 and L-ascorbate, a factor essential for collagen synthesis, on the synthesis and distribution of proteoglycans were examined using murine osteoblast-like MC3T3-E1 cells. TGF-beta 1 stimulated the synthesis of proteoglycans in MC3T3-E1 cells. Among various proteoglycans, the synthesis of decorin was preferentially enhanced by TGF-beta 1, and the effect was more pronounced on secreted decorin compared to that associated with the cell/matrix layer. TGF-beta 1 also stimulated the initiation and elongation of the dermatan sulfate glycosaminoglycan chain, resulting in a larger molecular size of decorin. TGF-beta 1 influenced the synthesis of a heparan sulfate proteoglycan only slightly. L-ascorbate had no effect on the synthesis of proteoglycans, but increased those associated with the cell/matrix layer. Furthermore, when L-ascorbate was added to the culture along with TGF-beta 1, the percentage of proteoglycans associated with the cell/matrix layer increased from 25.8 +/- 1.0 to 41.0 +/- 0.5%. These data demonstrate that TGF-beta 1 markedly stimulates the synthesis of proteoglycans, especially decorin, mainly as a secreting form, that the accumulation of decorin into matrix is enhanced by L-ascorbate, and that the effects of TGF-beta 1 and L-ascorbate are additive.(ABSTRACT TRUNCATED AT 250 WORDS)

Proliferation of osteoblast-like MC3T3-E1 cells was minimal in serum-free Eagle's minimum essential medium (MEM) but was enhanced by about 3.5-fold in serum-free alpha-modification of MEM (alpha-MEM). By adding back each of the extra constituents present in alpha-MEM to MEM, it was found that ascorbic acid was responsible for the sustained proliferation of MC3T3-E1 cells without serum. Ascorbic acid also stimulated the synthesis of collagen and increased the hydroxyproline content of MC3T3-E1 cell cultures markedly. Inhibitors of collagen synthesis, L-azetidine-2-carboxylic acid, cis-4-hydroxyproline, and 3,4-dehydroproline, almost completely eliminated the stimulatory effect of ascorbic acid on DNA synthesis of MC3T3-E1 cells. The dose response of the effect of L-azetidine-2-carboxylic acid on the hydroxyproline content closely paralleled that on DNA synthesis of MC3T3-E1 cells. Furthermore, a 10 times higher concentration of proline, which competes with L-azetidine-2-carboxylic acid for the incorporation into procollagen molecules, reversed the inhibition of DNA synthesis by L-azetidine-2-carboxylic acid. These results are consistent with the assumption that the stimulatory effect of ascorbic acid on the proliferation of MC3T3-E1 cells is mediated through its effect on the synthesis of collagen or some related protein. Furthermore, a fibronectin attachment peptide, GRGDTP, that competes with matrix proteins for specific binding to cell surface adhesion receptors also inhibited the stimulation of proliferation by ascorbic acid almost completely.(ABSTRACT TRUNCATED AT 250 WORDS)

To estimate the response to hormone replacement therapy (HRT) by bone metabolic markers, 36 patients with postmenopausal osteoporosis or osteopenia were studied to assess the correlation between percent baseline changes in lumbar bone mineral density (BMD) after 12 months and those in various bone metabolic markers after 3, 6, and 12 months of HRT. All the patients were treated with 0.625 mg of conjugated estrogen and 2.5 mg of medroxyprogesterone per day and continued for 12 months. BMD was significantly increased up to 4.19 +/- 0.87% after 6 months and 4.93 +/- 1.27% after 12 months of HRT (p = 0.0001 by analysis of variance). In accordance with this, changes in the levels of osteocalcin (p = 0.041), alkaline phosphatase (p = 0.0001), N-terminal osteocalcin (p = 0.0001), urinary excretion of pyridinoline/Cr (p = 0.0001), and deoxypyridinoline/Cr (p = 0.0001) were significantly decreased, respectively. Among these bone metabolic markers, only the change in the serum N-terminal osteocalcin at 3 months (r = 0.557, p = 0.0022), at 6 months (r = 0.470, p = 0.0184), and at 12 months (r = 0.545, p = 0.0061) significantly correlated with the change in BMD 12 months after HRT. The elution profiles of immunoreactive osteocalcin-related molecules in serum fractionated by reverse-phase high performance liquid chromatography revealed that the N-terminal fragment as well as the intact osteocalcin molecule decreased after 3 months of HRT. These results demonstrate that N-terminal osteocalcin is a suitable predictor for estimating good responders to HRT in postmenopausal women.

Transforming growth factor (TGF)-beta is known to promote tumor invasion and metastasis. Although bone morphogenetic proteins (BMPs), members of the TGF-beta family, are expressed in a variety of human carcinoma cell lines, their roles in tumor progression have not been fully clarified. In this study, we sought to determine the roles of BMPs in the progression of breast cancer bone metastasis using human breast cancer samples and a mouse xenograft model. Immunohistochemical analysis of samples from breast cancer patients as well as a mouse xenograft model of MDA-231-D, highly metastatic human breast cancer cells, revealed phospho-Smad2 and phospho-Smad1/5/8 staining in the nuclei of cancer cells in primary tumor and/or bone metastasis. Using a functional in vivo bioluminescence imaging system, we showed that TGF-beta- and BMP-induced transcriptional pathways are active in bone metastatic lesions in vivo. In addition, both TGF-beta3 and BMP-2 promoted the motility and invasiveness of the MDA-231-D cells in vitro. Moreover, expression of dominant-negative receptors for TGF-beta and/or BMPs in the MDA-231-D cells inhibited invasiveness in vitro and bone metastasis in the xenograft model. These results suggest that BMPs as well as TGF-beta promote invasion and bone metastasis of breast cancer.

It has been shown that in the mouse colon 26 tumor model, tumors grown in the subcutis (subcutis colon 26) caused early onset of cachectic syndromes, whereas those in the liver (liver colon 26) did not. Both interleukin (IL)-6 and parathyroid hormone-related protein (PTHrP) were involved in the development of cachectic syndromes in this tumor model. However, whether expression of PTHrP and IL-6 is differently regulated in the tumor microenvironment is unclear. In the present study, culturing the colon 26 cells under different conditions in vitro revealed that IL-6 production was increased by monolayer culture under a low-glucose condition but not by spheroid culture. In contrast, PTHrP production was increased by spheroid culture but not by monolayer culture, even under a low-glucose condition. Gene expression profiling revealed that the expression of cyclooxygenase (COX)-2 was up-regulated in both subcutis colon 26 and spheroid cultures, and that COX-2 inhibitor NS-398 suppressed PTHrP production in spheroid cultures. Furthermore, administration of NS-398 decreased the PTHrP level without affecting the tumor growth in mice bearing subcutis colon 26. These results demonstrate that production of PTHrP and IL-6 largely depends on the microenvironments in which tumors are developed or metastasized and that up-regulation of COX-2 in a necrobiotic environment leads to PTHrP production, thereby causing cachectic syndromes. (Cancer Sci 2007; 98: 1563–1569)

As a marker for bone formation, bone Gla protein (BGP) levels in the circulation have been measured in clinical research and management for metabolic bone diseases. We evaluated the clinical availability of a newly developed two-site immunoradiometric assay (IRMA) for human BGP and determined the serum BGP concentrations using this methodology in patients with abnormal calcium metabolism including those with end-stage renal disease undergoing maintenance dialysis. A cross-reactivity test revealed that this assay system specifically recognizes intact molecules (1-49) of BGP and excludes fragments of the molecules (1-19, 12-33, 23-33). Serum BGP levels in dialysis patients were positively correlated with those by conventional radioimmunoassay (RIA) (r = 0.918, p less than 0.00001, n = 37) as well as normal individuals (r = 0.935, p less than 0.0001, n = 16). However, the levels of BGP determined by IRMA were estimated to be significantly lower than those by RIA (23.6 +/- 9.8 vs. 29.6 +/- 9.1 ng/ml, p less than 0.00001). These results suggest that this IRMA system, with a rapid and easy procedure, excludes fragment forms of BGP in the circulation, which are found in uremic sera and probably attributed to increased bone resorption. Further studies are needed to ensure that serum intact BGP levels mainly reflect BGP production in osteoblasts, particularly in end-stage renal disease.

Differentiation of committed osteoblasts is controlled by complex activities involving signal transduction and gene expression, and Runx2 and Osterix function as master regulators for this process. Recently, CCAAT/enhancer-binding proteins (C/EBPs) have been reported to regulate osteogenesis in addition to adipogenesis. However, the roles of C/EBP transcription factors in the control of osteoblast differentiation have yet to be fully elucidated. Here we show that C/EBP homologous protein (CHOP; also known as C/EBPzeta) is expressed in bone as well as in mesenchymal progenitors and primary osteoblasts. Overexpression of CHOP reduces alkaline phosphatase activity in primary osteoblasts and suppresses the formation of calcified bone nodules. CHOP-deficient osteoblasts differentiate more strongly than their wild-type counterparts, suggesting that endogenous CHOP plays an important role in the inhibition of osteoblast differentiation. Furthermore, endogenous CHOP induces differentiation of calvarial osteoblasts upon bone morphogenetic protein (BMP) treatment. CHOP forms heterodimers with C/EBPbeta and inhibits the DNA-binding activity as well as Runx2-binding activity of C/EBPbeta, leading to inhibition of osteocalcin gene transcription. These findings indicate that CHOP acts as a dominant-negative inhibitor of C/EBPbeta and prevents osteoblast differentiation but promotes BMP signaling in a cell-type-dependent manner. Thus, endogenous CHOP may have dual roles in regulating osteoblast differentiation and bone formation.

The purpose of this study was to assess whether a nutritional supply of calcium (Ca) could be substituted for alfacalcidol (ALF) administration in preventing bone loss due to estrogen deficiency. Female Wistar-Imamichi rats (8 months old) were ovariectomized (OVX) or sham-operated. OVX rats received ALF administration (0.025, 0.5, or 0.1 microg/kg, p.o., 5 times a week) with standard rodent chow [Ca 1.2%, phosphorus (P) 1.04%], a Ca-enriched diet containing 2%, 4%, or 6% Ca (Ca/P ratio of 2, 4, and 6, respectively), or a Ca/P-enriched diet (Ca/P ratio of 1.2). After 12 weeks of treatment, all rats were killed to harvest the spine, serum, and urine samples. Neither the ALF treatment nor the Ca supplement caused hypercalcemia. In the spine, ALF prevented decreases in bone mineral density (BMD) and compressive strength of lumbar spine induced by OVX. Micro-computed tomographic analysis confirmed that ALF significantly improved the trabecular bone pattern factor and the structure model index and suppressed bone destruction. In contrast, of particular interest, high-dose Ca administration did not have marked effects on bone fragility. Also, when both Ca and P were administered in high doses, BMD and mechanical strength decreased dose-dependently, urinary P excretion significantly increased, and serum parathyroid hormone level increased. Together, it is difficult to adjust the Ca supply through diet alone without disrupting the balance between serum Ca and P levels. Consequently, we conclude that ALF is beneficial for the treatment of osteoporosis, which is not achieved by the use of a Ca supplement.

Although active vitamin D drugs have been used for the treatment of osteoporosis, how the vitamin D receptor (VDR) regulates bone cell function remains largely unknown. Using osteoprotegerin-deficient mice, which exhibit severe osteoporosis due to excessive receptor activator of NF-kappaB ligand/receptor activator of NF-kappaB (RANKL/RANK) stimulation, we show herein that oral treatment of these mice with 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3] inhibited bone resorption and prevented bone loss, suggesting that VDR counters RANKL/RANK signaling. In M-CSF-dependent osteoclast precursor cells isolated from mouse bone marrow, 1alpha,25(OH)2D3 potently and dose-dependently inhibited their differentiation into multinucleate osteoclasts induced by RANKL. Among signaling molecules downstream of RANK, 1alpha,25(OH)2D3 inhibited the induction of c-Fos protein after RANKL stimulation, and retroviral expression of c-Fos protein abrogated the suppressive effect of 1alpha,25(OH)2D3 on osteoclast development. By screening vitamin D analogs based on their c-Fos-suppressing activity, we identified a new analog, named DD281, that inhibited bone resorption and prevented bone loss in ovariectomized mice, more potently than 1alpha,25(OH)2D3, with similar levels of calcium absorption. Thus, c-Fos protein is an important target of the skeletal action of VDR-based drugs, and DD281 is a bone-selective analog that may be useful for the treatment of bone diseases with excessive osteoclastic activity.

Nude rats bearing the LC-6-JCK human lung cancer xenograft displayed cancer-associated wasting syndrome in addition to humoral hypercalcemia of malignancy. In these rats, not only PTHrP but also several other human proinflammatory cytokines, such as IL-6, leukemia-inducing factor, IL-8, IL-5 and IL-11, were secreted to the bloodstream. Proinflammatory cytokines induce acute-phase reactions, as evidenced by a decrease of serum albumin and an increase in alpha1-acid glycoprotein. Tumor resection abolished the production of proinflammatory cytokines and improved acute-phase reactions, whereas anti-PTHrP antibody affected neither proinflammatory cytokine production nor acute-phase reactions. Nevertheless, tumor resection and administration of anti-PTHrP antibody similarly and markedly attenuated not only hypercalcemia but also loss of fat, muscle and body weight. Body weight gain by anti-PTHrP antibody was associated with increased food consumption; increased body weight from anti-PTHrP antibody was observed when animals were freely fed but not when they were given the same feeding as those that received only vehicle. Furthermore, nude rats bearing LC-6-JCK showed reduced locomotor activity, less eating and drinking and low blood phosphorus; and anti-PTHrP antibody restored them. Although alendronate, a bisphosphonate drug, decreased blood calcium, it affected neither locomotor activity nor serum phosphorus level. These results indicate that PTHrP represses physical activity and energy metabolism independently of hypercalcemia and proinflammatory cytokine actions and that deregulation of such physiologic activities and functions by PTHrP is at least in part involved in PTHrP-induced wasting syndrome.

Fibroblast growth factor-23 (FGF-23), a novel phosphate-regulating factor, was elevated in hypophosphatemic patients with X-linked hypophosphatemic rickets/osteomalacia and also in patients with chronic kidney disease. These observations suggested the pathophysiological importance of FGF-23 on phosphate homeostasis. However, regulation of FGF-23 production is still unclear. We investigated effects of both dietary phosphorus and 1alpha,25-dihydroxyvitamin D(3) (1alpha,25(OH)(2)D(3)) on circulating FGF-23 in vivo Administration of. 1alpha,25(OH)(2)D(3) dose-dependently increased serum FGF-23 in thyroparathyroidectomized rats without correlating with serum inorganic phosphorus or serum parathyroid hormone. On the other hand, vitamin D receptor null mice had very low serum FGF-23 and did not respond to the 1alpha,25(OH)(2)D(3) administration. These observations suggested 1alpha,25(OH)(2)D(3) directly or indirectly regulates circulating FGF-23. Serum FGF-23 had a strong correlation with serum inorganic phosphorus controlled by dietary phosphorus in 5/6 nephrectomized rats. High phosphate diet elicited a 5-fold increase in serum FGF-23 compared with sham-operated rats, whereas serum FGF-23 did not correlate with serum calcium or serum creatinine in 5/6 nephrectomized rats. Administration of 1alpha,25-dihydroxyvitamin D(3) also elicited a severalfold increase in serum FGF-23 in the uremic rats. Taken together, this shows that both serum phosphorus and 1alpha,25(OH)(2)D(3) regulate circulating FGF-23 independent of each other. Therefore, we proposed there was a feedback loop existing among serum phosphorus, 1alpha,25(OH)(2)D(3), and FGF-23, in which the novel phosphate-regulating bone-kidney axis integrated with the parathyroid hormone-vitamin D(3) axis in regulating phosphate homeostasis.

ik3-2 is a close relative to ik3-1/Cables, an associator with cdk3 and cdk5. ik3-1/Cables has been identified to be a candidate tumor suppressor for colon and head/neck cancers. In agreement, it has been pointed out that ik3-1/Cables is a regulator for both p53- and p73-induced apoptosis [J. Biol. Chem. 277 (2002) 2951] although ectopic expression of ik3-1/Cables does not induce apoptosis. Here we show that adenovirus-mediated overexpression of ik3-2 results in apoptosis of p53-intact U2OS cells. ik3-2 binds to p53 in vivo and ectopic coexpression of ik3-2 enhances apoptosis induced by adenovirus-mediated expression of p53. Furthermore, ectopic expression of ik3-2 results in apoptosis of primary p53/Mdm2- and p53/ARF-null mouse embryo fibroblasts, indicating that ik3-2-induced apoptosis is partially p53-independent. Both the highly conserved C-terminal cyclin box-homologous domain (ik3-2-C) and the N-terminal region consisting of 70 amino acids (ik3-2-N) are responsible for ik3-2-mediated enhancement of p53-induced apoptosis. In contrast, ik3-2-induced p53-independent apoptosis is mediated through ik3-2-N. We thus identified ik3-2 as a proapoptotic factor involved in both p53-mediated and p53-independent apoptotic pathways.

Combined disruption of the ARF gene and the p53 gene causes mouse predisposition to tumors of a wider variety and at a higher frequency than disruption of the p53 gene, indicating that the ARF gene has p53-independent anti-tumor function in addition to p53-dependent function. Coincidentally with this notion, ectopic expression of the p19(ARF) induces apoptosis for wild-type mouse embryo fibroblasts which have been immortalized by introduction of the SV40 virus genome (SV40-MEFs). The protein expression levels of p53, p21(Cip1), and Bax were not upregulated by ectopic expression of p19(ARF) in SV40-MEFs, indicating that expression of p19(ARF) induced apoptosis through p53-independent pathways in this system. Ectopic expression of p19(ARF) induced prominent apoptosis even in SV40-Bak-/-MEFs. In contrast, expression of p19(ARF) induced only a very low grade of apoptosis in Bax-/- or Bax-/-/Bak-/-SV40-MEFs. Remarkable attenuation of p19(ARF)-induced apoptosis by disruption of the Bax gene thus leads to the conclusion that Bax plays a major role in p53-independent apoptosis induced by p19(ARF).

Parathyroid hormone-related protein (PTHrP) plays a central role in humoral hypercalcemia of malignancy (HHM), which is one of the most frequent paraneoplastic syndromes. PTHrP produced by the tumor acts through a common PTH/PTHrP receptor to promote bone resorption, inhibit calcium excretion from the kidney, and induce hypercalcemia. Patients with HHM often develop cachexia associated with typical symptoms such as anorexia, malaise, nausea, constipation, polyuria, polydipsia, and confusion. The etiology of the cachexia is not fully understood but is thought to be caused by hypercalcemia and various cytokines such as interleukin-6, tumor necrosis factor-alpha, leukemia inhibitory factor, and others. In this study, we investigated the role of PTHrP in hypercalcemia and cachexia in HHM by using humanized anti-PTHrP antibody. A mouse monoclonal antibody that binds to PTHrP amino acid sequence 1-34 and inhibits PTHrP function has been humanized to create a specific and potent agent for the treatment of patients with HHM. The mouse monoclonal antibody has been shown to have antihypercalcemic activity against nude mice bearing human tumors. Because a mouse antibody is highly immunogenic in human patients, the complementarity-determining regions from the mouse antibody were grafted into a human antibody. The resulting humanized antibody specifically recognizes PTHrP(1-34) and neutralizes PTHrP functions in vitro and in vivo. The humanized anti-PTHrP antibody was administered intravenously to HHM model animals bearing tumors such as LC-6 human lung carcinoma. These animals showed symptoms similar to those of patients with HHM (eg, hypercalcemia and cachexia). The humanized anti-PTHrP antibody-treated animals responded with normalization of blood ionized calcium level through an improvement of bone metabolism and calcium excretion. Moreover, the treated animals also showed an improvement in body weight, ultromotivity, metabolic alkalosis, food consumption, water intake, serum phosphorus, and renal function. Consequently, the humanized antibody-treated animals experienced complete resolution of hypercalcemia and cachexia. These results suggest that the humanized antibody would be an effective and beneficial agent for patients with HHM, and that PTHrP is a major pathogenetic factor of hypercalcemia and cachexia in patients with HHM.

Sentinel node (SN) biopsy is a promising replacement for standard axillary lymph node dissection for the staging of early breast cancer, and various techniques have been studied to identify SNs with dye or radioactive colloid. This study assesses the effect of the dose of radioactivity and the time before biopsy in order to set standards for the use of 99mTc-rhenium sulphide for the detection of SNs in breast cancer patients. Sixty patients with stage T1-2 N0 M0 breast cancer underwent SN biopsy, which was immediately followed by standard axillary dissection to confirm the SN results. For SN biopsy, 99mTc-rhenium colloid was injected peritumorally. A 1 day (morning injection and afternoon surgery) or 2 day (day before afternoon injection and morning surgery) protocol was applied. A dose-finding study was performed simultaneously using 7.4-37 MBq for the 1 day protocol and 37-74 MBq for the 2 day protocol. A scintigram was taken at 2 h for the 1 day protocol and 16 h for the 2 day protocol. After the injection of blue dye, SN biopsy was performed with a gamma probe, followed by standard axillary node dissection. The radiation exposure received by the surgical team during the operation was monitored. Histopathological comparison between SNs and axillary nodes was performed. Patient characteristics that might affect the radiocolloid uptake by SNs were assessed. SNs were identified in all patients regardless of the dose or administration protocol used. Two patients showed false negative pathological SN results, and the negative predictive value was 96% and the positive predictive value was 100%. In addition, radiation exposure to the surgical team and the amount of radioactive surgical waste were low, especially at lower doses. Two groups of patient characteristics were related to SN uptake. One was the body mass index (BMI) and the other was the age or menopausal status. Patients with a larger BMI tended to take up a smaller amount of 99mTc colloid. Older or post-menopausal patients showed lower SN uptake. 99mTc-rhenium sulphide colloid is an efficient radiopharmaceutical for SN detection. Both 1 day and 2 day protocols have equally good efficacy, and the recommended dose is 7.4 MBq for the 1 day protocol and 37 MBq for the 2 day protocol. Patients with larger BMI and older or post-menopausal patients tend to take up less 99mTc colloid.

The mechanism of transcriptional repression by nuclear hormone receptors, especially in the presence of the ligands, is largely unknown. We previously reported that 1,25-dihydroxyvitamin D(3) (1,25 vitamin D3) inhibited expression of the parathyroid hormone-related polypeptide (PTHrP) gene through the interaction between the liganded monomeric vitamin D receptor (VDR) and the negative DNA element in the PTHrP gene (nVDRE(RP)). In this study, we employed chromatin immunoprecipitation (ChIP) assay and confirmed that 1,25 vitamin D3 recruited DNA-dependent protein kinase (DNA-PKcs) to the chromatinized nVDRE(RP). Conversely, the regulatory subunits of DNA-PK were associated with the nVDRE(RP) sequences only when 1,25 vitamin D3 was absent. VDR was constitutively associated with these chromatinized nVDRE(RP) sequences. Furthermore, DNA-PKcs could phosphorylate VDR in vitro. We raise a possibility that a conformational change of VDR through its phosphorylation mediated by DNA-PKcs underlies the mechanism of gene repression by 1,25 vitamin D3-bound VDR.

The use of bisphosphonates for skeletal metastasis of breast cancer is now well established. Although clinical judgement for treating skeletal metastasis is based on symptoms and imaging studies, accurate or quantitative means are few. Various bone metabolic markers have been developed and these were evaluated in patients with metastasis to bone. Bone metabolic markers, especially resorption markers, have been shown to be a good tool for the monitoring the response to therapy for skeletal metastasis. This is also true for bisphosphonate treatment for skeletal metastasis. Bone metabolic markers are produced by different mechanisms. There are some different classes of resorption markers; tartrate-resistant acid phosphatase (TRAP) is secreted by osteoclast, N- and C-terminal cross-linking telopeptide of type I collagen (NTx and CTx) are the degradation the products of type I collagen, mainly produced by cathepsin K, and pyridinoline cross-linked carboxyl-terminal telopeptides of type I collagen (I CTP) is also a degradation product of type I collagen, by matrix metalloproteases. Even though bone resorption markers are a good tool to monitor response to bisphosphonate therapy, there remains the question of which class of bone resorption markers is best suited to the task.

A trial on the long-term administration of 1,25-dihydroxy-22-oxavitamin D(3) (22-oxacalcitoriol, OCT) was conducted among 124 patients with chronic renal failure on maintenance haemodialysis (HD) complicated with secondary hyperparathyroidism (2HPT). In the trial, OCT was administered three times weekly for 26 weeks subsequent to a 26-week pre-trial. As a result, intact-parathyroid hormone (PTH) levels fell significantly after the start of administration and, at the end of the trial, PTH was decreased by over 30% in 51.6% (64/124) of the patients, and the levels of bone metabolism markers such as alkaline phosphatase (ALP), bone ALP, and tartrate-resistant acid phosphatase (TRACP) were significantly decreased compared with those at the start of administration, suggesting a correction of high-turnover bone disease. Serum calcium (Ca) levels rose significantly following OCT administration, but were successfully maintained within a physiological level. Hypercalcaemia, which was diagnosed in 33.1% of patients, was found to resolve or ameliorate immediately after the withdrawal or dose reduction of OCT. OCT can be administered for as long as 1 year without any major problems other than hypercalcaemia. The final doses ranged from 2.5 to 20.0 microg/HD, and the optimal dose varied among patients depending on the intact-PTH and adjusted serum Ca levels. These results suggest that OCT is a highly effective drug for the suppression of PTH levels in 2HPT, and is an overall safe drug if the dosage is adjusted for serum Ca and intact-PTH levels. This study confirmed that the long-term (1-year) administration of OCT is very useful for the treatment of 2HPT.

p19(ARF) is a potent tumor suppressor. By inactivating Mdm2, p19(ARF) upregulates p53 activities to induce cell cycle arrest and sensitize cells to apoptosis in the presence of collateral signals. It has also been demonstrated that cell cycle arrest is induced by overexpressed p19(ARF) in p53-deficient mouse embryonic fibroblasts, only in the absence of the Mdm2 gene. Here, we show that apoptosis can be induced without additional apoptosis signals by expression of p19(ARF) using an adenovirus-mediated expression system in p53-intact cell lines as well as p53-deficient cell lines. Also, in primary mouse embryonic fibroblasts (MEFs) lacking p53/ARF, p53-independent apoptosis is induced irrespective of Mdm2 status by expression of p19(ARF). In agreement, p19(ARF)-mediated apoptosis in U2OS cells, but not in Saos2 cells, was attenuated by coexpression of Mdm2. We thus conclude that there is a p53-independent pathway for p19(ARF)-induced apoptosis that is insensitive to inhibition by Mdm2.

Currently, imaging techniques are the leading methods used to diagnose of metastasis to bone. However, these techniques are expensive, expose patients to toxic and radioactive compounds, and monitor response to treatment poorly; these drawbacks have prompted the search for alternative screening methods. Therefore, bone metabolic markers have been evaluated as possible methods to diagnose and monitor the development and progression of metastatic bone disease. Although bone metabolic markers are often grouped as either resorption or formation markers, studies have revealed that each marker has its own biologic meaning and clinical relevance. Recent milestones in the use of bone metabolic markers as screening methods for metastatic bone disease and as evaluation methods for treatment response are shown in the following lists. 1. Bone metabolic marker measurements provide insight into mechanisms of metastasis to bone. 2. Although promising data have been reported, bone metabolic markers are not yet considered to be reliable screening methods for metastasis to bone. 3. Bone metabolic markers are reliable indicators of response to both conventional and bisphosphonate therapies. 4. Preliminary results indicate bone metabolic markers might be an independent prognostic factor in patients whose tumors metastasize to bone. 5. New or refined assays for bone metabolic markers are expected to improve the sensitivity and specificity of bone metabolic marker use in diagnosing and monitoring metastasis to bone.

Although active vitamin D is used in certain countries for the treatment of osteoporosis, the risk of causing hypercalcemia/hypercalciuria means that there is only a narrow therapeutic window, and this has precluded worldwide approval. The results of our previous animal studies have suggested that the therapeutic effect of active vitamin D on bone loss after estrogen deficiency can be dissociated at least partly from its effect of enhancing intestinal calcium absorption and suppressing parathyroid hormone (PTH) secretion. To test this, we compared the effects of ED-71, a hydroxypropoxy derivative of 1alpha,25-dihydroxyvitamin D3, with orally administered alfacalcidol, on bone mineral density (BMD) and the bone remodeling process as a function of their effects on calcium metabolism and PTH, in a rat ovariectomy (ovx) model of osteoporosis. ED-71 increased bone mass at the lumbar vertebra to a greater extent than alfacalcidol, while enhancing calcium absorption (indicated by urinary calcium excretion) and decreasing serum PTH levels to the same degree as alfacalcidol. ED-71 lowered the biochemical and histological parameters of bone resorption more potently than alfacalcidol, while maintaining bone formation markers. These results suggest that active vitamin D exerts an antiosteoporotic effect by inhibiting osteoclastic bone resorption while maintaining osteoblastic function, and that these anticatabolic/anabolic effects of active vitamin D take place independently of its effects on calcium absorption and PTH. The demonstration that ED-71 is more potent in these properties than alfacalcidol makes it an attractive candidate as an antiosteoporotic drug.

Although skeletal metastasis is a common complication associated with prostate cancer, assessment of the response of osseous metastasis to therapy is still difficult. To remedy this situation a study of bone formation metabolic markers gaps in patients with prostate cancer and osseous metastasis was done to determine these gaps as clinical markers for the efficacy of therapy against osseous metastasis.The activities of the bone formation markers amino terminal propeptide of type I procollagen (PINP), bone alkaline phosphatase (BAP) and osteocalcin (OC) were measured in 57 and 60 patients with prostate cancer without and with osseous metastasis, respectively. Of the 60 patients with osseous metastasis serial measurements were performed in 31. The status of osseous metastasis at the time of marker measurement was categorized, and the BAP-to-PINP, OC-to-PINP and OC-to-BAP ratios were calculated.The BAP-to-PINP ratio did not change. However, the OC-to-PINP and OC-to-BAP ratios were significantly different depending on osseous metastasis status. The ratios were high in patients with no osseous metastasis or with metastasis shrinkage (improvement) but low in those in whom metastases were growing and/or spreading (progression). Cutoff levels of OC-to-PINP and OC-to-BAP ratios were determined by comparing ratios with clinical judgment on the response to treatment in all patients. When these cutoffs were applied to the 31 patients, the OC-to-PINP ratio agreed with clinical judgment in 29 (94%) and the OC-to-BAP ratio agreed in 28 (90%).Different bone formation markers seem to be sensitive clinical markers for judging the response to therapy of prostate cancer metastasized to bone.

Background/Aims: Although the PTH-suppressive effect of intravenous calcitriol has already been demonstrated by various studies, the precise dose-response to calcitriol has not been fully determined for uremic secondary hyperparathyroidism (2HPT). In order to investigate in detail the dose-response of intravenous calcitriol and the adequate initial dose against 2HPT, a randomized prospective double-blind study was conducted. Method: One-hundred and sixty-two patients with 2HPT undergoing hemodialysis three times per week were randomly assigned to four calcitriol (Ro21-5535) treatment groups, 0 (placebo), 1, 1.5 or 2 µg. Calcitriol or placebo was given intravenously after each dialysis for 12 weeks under double-blind conditions. Results: Calcitriol dose-dependently reduced both intact-PTH and high-sensitivity assay mid-terminal (HS)-PTH levels. The rate of per-week change in intact-PTH was 0.0% in the placebo group, –7.8% in the 1-µg group, –18.9% in the 1.5-µg group and –24.1% in the 2-µg group. Calcitriol dose-dependently increased the rate of increase in serum Ca adjusted by albumin level. The per-week increases in adjusted serum Ca were –0.01, 0.08, 0.23 and 0.35 mg/dl in the placebo, 1-, 1.5- and 2-µg groups, respectively. Although the degree of PTH suppression was correlated with the adjusted serum Ca increase, by-patients investigation revealed that the number of patients with suppression of PTH despite of no or slight elevation of adjusted serum Ca level was largest in the 1-µg group among the three calcitriol groups. Conclusion: Intravenous calcitriol was found to have a clear dose-dependent effect on PTH reduction in patients with 2HPT, and the appropriate initial dose of this agent was determined to be 1 µg per dialysis session.

A case of transient splenic accumulation of the bone-seeking agent Tc-99m HMDP is reported. This effect was caused by transient megaloblastic anemia induced by 5-fluorouracil chemotherapy. The extent of splenic uptake reflected the development and severity of megaloblastic anemia. The mechanism of splenic accumulation is thought to be similar to transient iron deposition in the spleen by megaloblastic anemia.

Background. Bisphosphonates are bone resorption inhibitors which are effective in the treatment of diseases of increased bone turnover, such as hypercalcemia of malignancy and osteolytic bone metastasis. The safety and efficacy of incadronate, a third-generation bisphosphonate, were evaluated in breast cancer patients with bone metastases.

Androgens play an important role in the growth of prostate cancer, but the molecular mechanism that underlies development of resistance to antiandrogen therapy remains unknown. Cyclin E has now been shown to increase the transactivation activity of the human androgen receptor (AR) in the presence of its ligand dihydrotestosterone. The enhancement of AR activity by cyclin E was resistant to inhibition by the antiandrogen 5-hydroxyflutamide. Cyclin E was shown to bind directly to the COOH terminus portion of the AB domain of the AR, and to enhance its AF-1 transactivation function. These results suggest that cyclin E functions as a coactivator of the AR, and that aberrant expression of cyclin E in tumors may contribute to persistent activation of AR function, even during androgen ablation therapy.

The effects of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] and its analogue 22-oxa-1,25(OH)2D3 (22-oxacalcitriol) (OCT) on calcium and bone metabolism were examined in an animal model of hypercalcemia with continuous infusion of parathyroid hormone-related peptide (PTHrP), to determine whether active vitamin D could counteract the skeletal action of PTHrP in addition to its reported effect in suppressing the production of PTHrP in cancer cells. Parathyroid glands were removed from 8-week-old Sprague-Dawley rats to eliminate the confounding effects of endogenous PTH. Animals were then continuously infused with human PTHrP(1-34) at a constant rate via osmotic minipumps for 2 weeks, and at the same time treated orally or intravenously with OCT or 1,25(OH)2D3 four to nine times during the 2-week period. Under these conditions, OCT and, surprisingly, 1,25(OH)2D3 alleviated hypercalcemia in a dose-dependent manner. 1,25(OH)2D3 and OCT suppressed the urinary excretion of deoxypyridinoline, although they did not affect renal calcium handling, suggesting that the antihypercalcemic effect is attributable to the inhibition of bone resorption. These active vitamin D compounds also counteracted the effects of PTHrP at the proximal renal tubules, as reflected by a decrease in phosphate excretion. Histomorphometric analysis of bone revealed a dose-related decrease in parameters of bone resorption. These results suggest that 1,25(OH)2D3 as well as OCT has the potential to alleviate hypercalcemia, at least in part, through the inhibition of bone resorption in hypercalcemic rats with constant PTHrP levels. We propose that the main function of active vitamin D in high bone-turnover states is to inhibit bone resorption, and this may have important implications for the understanding of the role of active vitamin D in the treatment of metabolic bone diseases, such as osteoporosis.

Estrogen receptor (ER)-positive breast cancers initially respond well to estrogen ablation treatment but finally acquire refractoriness, the phenomenon that is a major clinical problem. Because some breast cancers synthesize estradiol (E2) and E2 synthesis is regulated by gonadotropins in normal ovaries, and because circulating gonadotropins are elevated in postmenopausal women and during estrogen ablation treatment, we hypothesized that gonadotropins might modulate estrogen synthesis/metabolism in breast cancer tissue as well. To test this possibility, MCF-7 cells were treated with dehydroepiandrosterone (DHEA) or human chorionic gonadotropin (hCG; ∼LH), each alone or in combination. Cell growth (3-day treatment) was assayed by the MTT method and estrogen synthesis (24-hour treatment) was measured using the ERE-luciferase reporter system. First, MCF-7 cell growth was stimulated by DHEA in a concentration-dependent manner with a maximal effect at 10–4M. Although hCG alone did not have a significant proliferative effect, hCG significantly and dose dependently stimulated MCF-7 cell growth in the presence of a submaximal concentration of DHEA (10–7 M). This stimulatory effect of DHEA and hCG was blocked by a pure antiestrogen ICI182,780 and an aromatase inhibitor, arimidex. Using MCF-7 cells transfected with the ERE-luciferase reporter system, hCG treatment was shown to increase ERE-mediated transcription. These results indicate that MCF-7 cells intrinsically converted DHEA into E2 upon hCG stimulation, then grew their own cells DHEA- and hCG-dependently. We conclude that gonadotropins can act on breast cancer cells and accelerate conversion of DHEA into estrogens, thereby stimulating growth of estrogen-dependent tumor cells. This phenomenon, at least in part, could explain: (1) an increased tissue concentration of E2 in postmenopausal breast cancer; (2) acquisition of hormone refractoriness during estrogen ablation treatment, and (3) the effectiveness of GnRH antagonist/superagonist in some postmenopausal breast cancer patients.

Background. The efficacy and cost-performance benefit of radionuclide bone scintigraphy in monitoring metastatic bone activity remain controversial. Bone metabolic markers are now expected to play a role in the diagnosis and follow-up of bone metastasis.

Bone scan flare seriously complicates evaluations of the therapeutic response of bone metastases. The value of bone metabolic markers in monitoring the therapeutic response for bone metastases in breast cancer was assessed. Twenty-three breast cancer patients with bone metastases treated by combined chemotherapy of cyclophosphamide, doxorubicin, and 5-fluorouracil (CAF) were monitored using bone scans; a bone resorption marker, pyridinoline cross-linked carboxy-terminal telopeptide of type I collagen (ICTP); a bone formation marker, bone-specific alkaline phosphatase (BAI-p); and a tumor-specific marker (CA15-3). Bone scans were performed before and 3 or 4 months after therapy. After CAF therapy, markers were measured monthly. As a control, the markers of nine patients without bone metastases who received adjuvant CAF therapy were also measured monthly. The therapeutic effect on bone metastases was assessed after the study. Five patients had progression of disease (PD), three had no change (NC), and 15 patients had partial responses (PR). Bone scan flare-up was seen in five PR patients. In patients who received adjuvant therapy, ICTP, BAI-p, and CA15-3 did not change. ICTP increased significantly in PD patients. ICTP did not increase in either NC or PR, including bone scan flare patients. BAI-p and CA15-3 did not show any discernible pattern among PD, PR, flare, and NC patients. Thus measuring ICTP could distinguish PD from NC or PR patients' responses to CAF therapy. This was true also for patients who showed bone scan flare-up. Measuring a bone resorption marker, ICTP, allows clinicians to monitor patients' responses to CAF therapy and may prevent prolonged ineffective therapy or unnecessary changes in therapy as a result of the flare phenomenon.

Hypercalcemia represents one of the important paraneoplastic syndromes affecting morbidity and mortality of cancer patients. We and others have demonstrated that vitamin D analogs with little calcemic activities suppress the transcription of the parathyroid hormone-related peptide (PTHrP) gene, a major humor responsible for cancer hypercalcemia, and thereby prevent the development of hypercalcemic syndrome. The present study was undertaken: to compare the therapeutic efficacy of a vitamin D analog, 22-oxa-1,25-dihydroxyvitamin D3 (OCT), and a bisphosphonate (disodium 3-amino-1-hydroxypropylidene-1,1-bisphosphonate pentahydrate [AHPrBP]), an inhibitor of osteoclastic bone resorption, on cancer-induced hypercalcemia; and to see if the effect could be enhanced by combination treatment, using a nude mouse model implanted with a human pancreas carcinoma (FA-6). After a single intravenous administration, OCT (5 microg/kg of body weight [BW]) was as effective as AHPrBP (10 mg/kg of BW) in lowering blood ionized calcium levels in tumor-bearing nude mice, and their combination further enhanced the therapeutic effect. Although AHPrBP lost its efficacy after repeated injections, OCT was still effective after the third administration. The therapeutic effect of OCT in cancer hypercalcemia was observed in four other human tumors, including another pancreas carcinoma (PAN-7), two squamous cell carcinomas of the lung (KCC-C1 and LC-6), and a squamous carcinoma of the pharynx (PHA-1), all of which elaborated PTHrP into the circulation. Treatment with OCT resulted in a decrease in circulating PTHrP levels by approximately 50% in two representative models. However, the mechanism underlying the antihypercalcemic effect of OCT seemed complex, involving inhibition of PTHrP production, suppression of excessive bone resorption, and an antitumor activity. OCT also markedly inhibited the body weight loss with tumor growth, while AHPrBP, which exhibited a similar antihypercalcemic effect, was less effective than OCT in preventing cachexia. The anticachectic activity of their combination did not exceed that of OCT alone, suggesting a hypercalcemia-dependent as well as an independent mechanism of cancer cachexia. It is concluded that OCT may be useful, either as a single agent or in combination with bisphosphonates, for the treatment of cancer-associated hypercalcemia and cachexia.

Purpose: We examined the correlation between bone metabolic markers and bone scintigraphy in prostatic cancer.Materials and Methods: Osteoblastic and osteoclastic markers, prostate specific antigen (PSA) and bone scans were investigated in 83 specimens from 70 patients with prostatic cancer, including 32 with and 38 without bone metastasis.Results: All markers except for osteocalcin were significantly greater in patients with than without bone metastasis. Pyridinoline cross-linked carboxyterminal telopeptide, an osteoclastic marker, reflected the extent of bone metastasis more accurately than PSA and other bone markers.Conclusions: Pyridinoline cross-linked carboxyterminal telopeptide might assist PSA and bone scintigraphy in monitoring metastatic bone activity of prostatic cancer.

To evaluate the effects of 1 alpha-hydroxyvitamin D3 (1 alpha(OH)D3), a series of clinical trials, preventive and therapeutic, were performed in an open label manner in women immediately after oophorectomy. The series included a total of 121 oophorectomized subjects, whose lumbar bone mineral density (L2-4BMD) was followed by the use of dual energy X-ray absorptiometry. (1) Preventive trial: 61 women who had undergone premenopausal bilateral oophorectomy, were divided into 3 groups (Group C: control; Group L: 0.25 micrograms 1 alpha (OH)D3/day; Group H: 0.50-0.75 micrograms 1 alpha (OH)D3/day). The changes in BMD and chemical indices were followed up for one year. (2) Therapeutic trial: the trial included 60 premenopausally oophorectomized subjects having L2-4BMD lower than the normal control level minus 1SD which has been reported in age-matched normal Japanese women. These subjects were divided into 3 groups and treated in the same way as in the preventive trial. In the preventive trial, L2-4BMD decreased by 8.2%, 6.5% and 4.5% in groups C, L and H, respectively, at 12 months of treatment, whereas in the therapeutic trial, L2-4BMD decreased by 3.6%, 3.2% and 0.8% in the groups C, L and H, respectively, at 12 months of treatment. In conclusion, 1 alpha(OH)D3 was found to be effective both to prevent the bone loss subsequent to bilateral oophorectomy and improve low bone mass after oophorectomy.

Background Bisphosphonates are powerful inhibitors of osteoclast-mediated bone resorption. They are effective in the treatment of Paget's disease of the bone, tumor-associated hypercalcemia, and osteoporosis. They are also used to treat metastatic bone disease. YM-175 is a new highly potent bisphosphonate. Bisphosphonates are also used as radiopharmaceuticals in bone scintigraphy. The data remain unclear as to whether or not the administration of large amounts of bisphosphonate interferes with the bone scintigraphy process.

Through the specific binding of a negative calcium-responsive element to its binding protein in response to extracellular Ca (Ca2+e), negative calcium-responsive element-bearing genes, such as the human parathyroid hormone gene, are negatively regulated by Ca2+e. The Ku antigen mediated negative gene regulation by Ca2+e by interacting with a redox factor protein, REF1. Although sequence-nonspecific DNA binding activity of the Ku antigen has been well characterized, the mechanism of its sequence-specific DNA binding remained obscure. Here, we report that the specific binding of the Ku antigen to another protein, REF1, leads to DNA-protein complex formation with a novel sequence specificity and thereby regulates gene expression.

Amyloid precursor protein (APP) has been shown to serve as a G(o)-coupled receptor in cell-free systems [Okamoto et al: J Biol Chem 1995;270:4205-4208]. However, it has not been known whether APP exerts intracellular signaling functions in living cells. In this study, we show that stimulation of APP by anti-APP antibody as well as by a mutation found in familial Alzheimer's disease results in activation of a specific set of mitogen-activated protein kinases in multiple vertebrate cells. We conclude that APP acts as a cell surface receptor of biological relevance that turns on specific Ser/Thr kinases, and suggest that the signaling function of APP is a potential target of familial Alzheimer's disease mutations.

Summary: Severe secondary hyperparathyroidism in chronic dialysis patients has been recently treated by supraphysiological concentration of calcitriol achieved through pulse therapy. However, there are many patients resistant to this therapy, who usually have larger parathyroid gland(s). to overcome this resistance, calcitriol was injected directly into the enlarged glands under ultrasonographic guidance. We injected 70–90% of the calculated gland volume of calcitriol solution (1 μg/mL) into the glands of 7 patients three times per week for 2 weeks. the parathyroid hormone (PTH) levels decreased significantly after 2 weeks of direct injections of calcitriol. Following a further 4 weeks of calcitriol pulse therapy, PTH levels remained suppressed and serum alkaline phosphatase activity and the volume of parathyroid glands also decreased. During the long-term follow up, five patients remained well controlled with calcitriol pulse therapy, while two patients needed ethanol injections to control hyperparathyroidism. Although we could not completely rule out a toxic effect of the vehicle, direct injection of calcitriol into parathyroid glands may be another treatment option for chronic dialysis patients. Our data further support the important role of resistance of parathyroid cells to calcitriol in the pathogenesis of parathyroid hyper function in uraemic patients.

The present study was undertaken to identify serum components other than vitamin D-binding proteins that bind to 1,25(OH)2 D3, and its analog. The binding rate of 1,25(OH)2 D3, 22-oxa-1,25(OH)2D3 (OCT) or 25(OH) D3 to total lipoprotein(TLP) represented 16.7%, 4.65%, and 3.11% of total counts added, respectively. Polyacrylamide gel electrophoresis of the TLP revealed that 1,25(OH)2 D3 and OCT were associated with LDL. The binding studies of OCT-bound LDL to the fibroblasts showed specific pathway to the cells mediated by LDL-receptor. These findings may have important implications in understanding the mechanisms of the diverse biological actions of 1,25(OH)2 D3 and in designing a novel delivery system for vitamin D analogs.

The efficacy and cost/performance benefit of radionuclide bone scintigraphy in monitoring metastatic bone activity remain controversial. Recently developed bone metabolic markers are expected to play an additional role in the diagnosis of bone metastasis. We measured osteoclastic and osteoblastic markers in 267 patients with breast cancer (100 with bone metastasis), 38 patients with prostatic cancer (25 with bone metastasis), 50 patients with lung cancer (12 with bone metastasis) and 33 patients with miscellaneous cancers (13 with bone metastasis) and compared the values in the presence and absence of bone metastasis. Bone metabolic markers, both osteoclastic and osteoblastic, increased significantly in patients with bone metastasis. In breast cancer (bone metastasis is mostly of the mixed type), osteoclastic markers were good in detecting bone metastasis. In prostatic cancer (bone metastasis is mostly osteoblastic), osteoclastic and osteoblastic markers were equally effective in detecting bone metastasis. In lung cancer (bone metastasis is mostly osteolytic), osteoclastic markers were elevated preferentially in bone metastasis. Over all, osteoclastic markers were more sensitive in the diagnosis of bone metastasis, and among osteoclastic markers, serum pyridionoline-cross-linked carboxyterminal telopeptide was the most efficient in both specificity (91.0%) and sensitivity (48.6%) for detecting bone metastasis.

Humoral hypercalcemia of malignancy (HHM) is a common paraneoplastic syndrome mediated by tumor-derived parathyroid hormone-related peptide (PTHRP), which bears structural and functional similarities to PTH. Thus the clinical features of HHM are very similar to those of primary hyperparathyroidism (1 degree HPT), a prototype of humoral hypercalcemia caused by PTH. On the other hand, HHM syndrome differs from 1 degree HPT in several aspects, including serum 1,25(OH)2D levels, acid-base balance, and bone remodeling process, the reason of which remains largely unknown. We approached these questions using a unique animal model of HHM, nude rats implanted with PTHRP-overproducing human carcinomas. In this review we will summarize the results and discuss the implications in understanding the disease mechanism.

The present study was undertaken to clarify the pharmacokinetics of 22-oxa-1,25-dihydroxyvitamin D3 (22-oxa-1,25-(OH)2D3, OCT), a vitamin D3 analogue with little calcemic activity, and its effect on the transcription of parathyroid hormone-related peptide (PTHRP) gene in nude mice bearing a human carcinoma (FA-6) associated with humoral hypercalcemia. FA-6 tumor expressed vitamin D receptor (VDR) mRNA, and its nuclear extract contained a specific and saturable 1,25-(OH)2D3 binding activity. Although [3H]OCT administered intravenously into FA-6 tumor-bearing nude mice was cleared from the circulation more rapidly than [3H]1,25-(OH)2D3, the uptake of [3H]OCT into the tumor tissue, relative to the radioactivity in the circulation, was greater than that of [3H]1,25-(OH)2D3. Intravenous or oral administration of OCT reduced the steady-state levels of PTHRP mRNA in FA-6 tumor, and nuclear run-off assays demonstrated that the effect of OCT on PTHRP gene expression occurred at a transcriptional level. RNase mapping analysis revealed that both upstream and downstream promoters of the human PTHRP gene were down-regulated by OCT. Finally, OCT exerted a preventive as well as therapeutic effect on cancer-associated hypercalcemia with a marked prolongation of the survival time in tumor-bearing animals. These results suggest that OCT is effectively taken up by a VDR-positive human carcinoma in vivo and has a therapeutic potential for cancer-associated hypercalcemia through suppression of PTHRP gene transcription.