Malignant melanoma and bone resorption

The cellular and humoral mechanisms accounting for osteolysis in skeletal metastases of malignant melanoma are uncertain. Osteoclasts, the specialised multinucleated cells that carry out bone resorption, are derived from monocyte/macrophage precursors. We isolated tumour-associated macrophages (TAMs) from metastatic (lymph node/skin) melanomas and cultured them in the presence and absence of osteoclastogenic cytokines and growth factors. The effect of tumour-derived fibroblasts and melanoma cells on osteoclast formation and resorption was also analysed. Melanoma TAMs (CD14+/CD51−) differentiated into osteoclasts (CD14−/CD51+) in the presence of receptor activator for nuclear factor κB ligand (RANKL) and macrophage-colony stimulating factor. Tumour-associated macrophage-osteoclast differentiation also occurred via a RANKL-independent pathway when TAMs were cultured with tumour necrosis factor-α and interleukin (IL)-1α. RT–PCR showed that fibroblasts isolated from metastatic melanomas expressed RANKL messenger RNA and the conditioned medium of cultured melanoma fibroblasts was found to be capable of inducing osteoclast formation in the absence of RANKL; this effect was inhibited by the addition of osteoprotegerin (OPG). We also found that cultured human SK-Mel-29 melanoma cells produce a soluble factor that induces osteoclast differentiation; this effect was not inhibited by OPG. Our findings indicate that TAMs in metastatic melanomas can differentiate into osteoclasts and that melanoma fibroblasts and melanoma tumour cells can induce osteoclast formation by RANKL-dependent and RANKL-independent mechanisms, respectively

Aberrant overexpression of an epithelial marker, 14-3-3σ, in a subset of hematological malignancies

<p>Abstract</p> <p>Background</p> <p>14-3-3σ is a p53-mediated cell-cycle inhibitor in epithelial cells. The expression of 14-3-3σ is frequently altered in cancers of epithelial origin associated with altered DNA methylation. Since its involvement in a non-epithelial tumor is unknown, we examined 14-3-3σ expression in patients with haematological malignancies.</p> <p>Methods</p> <p>We analyzed 41 hematopoietic cell lines and 129 patients with a variety of hematological malignancies for 14-3-3σ expression with real-time RT-PCR. We also examined protein levels by Western blot analysis and DNA methylation status of the 14-3-3σ gene by methylation-specific PCR analysis of bisulfite-treated DNA. In addition, mutations of p53 gene were identified by RT-PCR-SSCP analysis and the expression levels of 14-3-3σ were compared with those of other cell-cycle inhibitor genes, CDKN2A and ARF.</p> <p>Results</p> <p>The expression levels of 14-3-3σ mRNA in almost all cell lines were low and comparable to those in normal hematopoietic cells except for 2 B-cell lines. On the contrary, 14-3-3σ mRNA was aberrantly overexpressed frequently in mature lymphoid malignancies (30 of 93, 32.3%) and rarely in acute leukemia (3 of 35, 8.6%). 14-3-3σ protein was readily detectable and roughly reflected the mRNA level. In contrast to epithelial tumors, methylation status of the 14-3-3σ gene was not associated with expression in hematological malignancies. Mutations of p53 were identified in 12 patients and associated with lower expression of 14-3-3σ. The expression levels of 14-3-3σ, CDKN2A and ARF were not correlated with but rather reciprocal to one another, suggesting that simultaneous overexpression of any two of them is incompatible with tumor growth.</p> <p>Conclusion</p> <p>14-3-3σ, an epithelial cell marker, was overexpressed significantly in a subset of mature lymphoid malignancies. This is the first report of aberrant 14-3-3σ expression in non-epithelial tumors <it>in vivo</it>. Since the significance of 14-3-3σ overexpression is unknown even in epithelial tumors such as pancreatic cancers, further analysis of regulation and function of the 14-3-3σ gene in non-epithelial as well as epithelial tumors is warranted.</p

Development of a laser amplification system for the multi-pass Thomson scattering system for GAMMA 10/PDX

The multi-pass Thomson scattering (MPTS) system is a useful technique for increasing the Thomson scattering (TS) signal intensities and improving the TS diagnostic time resolution. The MPTS system developed in GAMMA 10/PDX has a polarization-based configuration with an image relaying system. The MPTS system has been constructed for enhancing the Thomson scattered signals for the improvement of measurement accuracy and the megahertz sampling time resolution. However, in the normal MPTS system, the MPTS signal intensities decrease with the pass number because of the damping due to the optical components. Subsequently, we have developed a new MPTS system with the laser amplification system. The laser amplification system can improve the degraded laser power after six passes in the multi-pass system to the initial laser power. For the first time worldwide, we successfully obtained the continued multi-pass signals after the laser amplification system in the gas scattering experiments

Functional Expression of the Extracellular Calcium Sensing Receptor (CaSR) in Equine Umbilical Cord Matrix Size-Sieved Stem Cells

The present study investigates the effects of high external calcium concentration ([Ca(2+)](o)) and the calcimimetic NPS R-467, a known calcium-sensing receptor (CaSR) agonist, on growth/proliferation of two equine size-sieved umbilical cord matrix mesenchymal stem cell (eUCM-MSC) lines. The involvement of CaSR on observed cell response was analyzed at both the mRNA and protein level.A large (>8 µm in diameter) and a small (<8 µm) cell line were cultured in medium containing: 1) low [Ca(2+)](o) (0.37 mM); 2) high [Ca(2+)](o) (2.87 mM); 3) NPS R-467 (3 µM) in presence of high [Ca(2+)](o) and 4) the CaSR antagonist NPS 2390 (10 µM for 30 min.) followed by incubation in presence of NPS R-467 in medium with high [Ca(2+)](o). Growth/proliferation rates were compared between groups. In large cells, the addition of NPS R-467 significantly increased cell growth whereas increasing [Ca(2+)](o) was not effective in this cell line. In small cells, both higher [Ca(2+)](o) and NPS R-467 increased cell growth. In both cell lines, preincubation with the CaSR antagonist NPS 2390 significantly inhibited the agonistic effect of NPS R-467. In both cell lines, increased [Ca(2+)](o) and/or NPS R-467 reduced doubling time values.Treatment with NPS R-467 down-regulated CaSR mRNA expression in both cell lines. In large cells, NPS R-467 reduced CaSR labeling in the cytosol and increased it at cortical level.In conclusion, calcium and the calcimimetic NPS R-467 reduce CaSR mRNA expression and stimulate cell growth/proliferation in eUCM-MSC. Their use as components of media for eUCM-MSC culture could be beneficial to obtain enough cells for down-stream purposes

Identification of a Second Kindred with Familial Hypocalciuric Hypercalcemia Type 3 (FHH3) Narrows Localization to a

CONTEXT: Familial hypocalciuric hypercalcemia (FHH) is a genetically heterogenous disorder that consists of three defined types, FHH1, FHH2, and FHH3 whose chromosomal locations are 3q21.1, 19p, and 19q13, respectively. FHH1, caused by mutations of the calcium-sensing receptor (CASR), occurs in more than 65% of patients, whereas the abnormalities underlying FHH2 and FHH3, which have each been described in single North American kindreds, are unknown. OBJECTIVE: The aim of this study was to determine the basis of FHH in a proband, who did not have CASR mutations, and her kindred. PATIENTS AND METHODS: The proband was a 43-yr-old woman who presented with a corrected serum calcium of 2.74 mmol/liter (normal = 2.15-2.55 mmol/liter), a serum PTH of 47 pg/ml (normal = 10-65 pg/ml), and a urinary calcium clearance:creatinine clearance of 0.006. She did not have a CASR mutation within the coding region and splice sites, and 24 members from three generations of her kindred were ascertained and investigated for serum abnormalities and cosegregation with polymorphic loci from chromosomes 3q21.1 and 19q13 using leukocyte DNA. RESULTS: Sixteen members were hypercalcemic with normal or elevated serum PTH concentrations and mild hypophosphatemia, features consistent with FHH3. Use of microsatellite and single nucleotide polymorphic loci from chromosome 19q13.3 demonstrated cosegregation with FHH in the kindred, with a peak LOD score = 5.98 at 0% recombination with D19S412. Analysis of recombinants mapped FHH to a 3.46-Mbp interval flanked centromerically by single nucleotide polymorphism rs1990932 and telomerically by D19S604. CONCLUSIONS: FHH3 may explain the calcium homeostasis disorder in those FHH patients who do not have CASR mutations