Human breast cancer cell lines and tissues express tartrate-resistant acid phosphatase (TRAP)

Tartrate-resistant acid phosphatase (TRAP) is expressed by osteoclasts, macrophages and dendritic cells. TRAP has been identified in a wide variety of tissues, however, its biological function is not fully understood. Serum TRAP is a marker of diseases involving excessive bone resorption including metastatic bone disease in breast cancer patients and can be used to monitor responses to treatment. Our aim in this study was to determine whether TRAP is expressed by human breast tumours. Four breast cancer cell lines were assayed for TRAP activity. MDA-MB-435, the most tumourigenic line, had an activity twofold higher than the other cell lines. Immunohistochemistry using a TRAP specific antibody confirmed that both cell lines and human breast tumours express TRAP. Expression was absent in benign tissues and abundant in more aggressive tumours. This work suggests that tumour derived TRAP contributes to the raised enzyme activity found in the serum of breast cancer patients.     

Parathyroid hormone related protein and hypercalcaemia in breast cancer.

OBJECTIVE--To see whether parathyroid hormone related protein has a humoral role in breast cancer. DESIGN--Plasma concentrations and tumour expression of parathyroid hormone related protein were determined (by two site immunoradiometric assay and immunohistochemistry respectively) in women with breast cancer and related to the presence of bone metastases and serum calcium concentrations. SUBJECTS--Plasma concentrations of parathyroid hormone related protein were measured in 57 women with early breast cancer without apparent bone metastases, 28 women with bone metastases, and 13 women with bone metastases and hypercalcaemia. Tissue positivity for parathyroid hormone related protein was determined retrospectively in 106 primary breast tumours from women without apparent bone metastases and 72 tumours from women with bone metastases, 25 of whom subsequently developed hypercalcaemia. RESULTS--Plasma parathyroid hormone related protein concentrations were detectable (greater than 0.23 pmol/l) in 12 (92%) of the 13 hypercalcaemic patients with bone metastases compared with 10 (36%) of the 28 normocalcaemic patients with bone metastases and five (9%) of the 57 normocalcaemic patients without bone metastases. Parathyroid hormone related protein concentrations were significantly higher in hypercalcaemic than normocalcaemic patients with bone metastases. Tumour staining was positive for parathyroid hormone related protein in 22 (88%) of the 25 primary breast cancers from patients with bone metastases. Tumour staining was positive for parathyroid hormone related protein in 22 (88%) of the 25 primary breast cancers from patients with bone metastases who later developed hypercalcaemia compared with 25 (53%) of the 47 from women in this group who remained normocalcaemic and 55 (52%) of the 106 early breast cancers from women without known metastases. CONCLUSION--Tumour derived parathyroid hormone related protein may have an important humoral role in hypercalcaemia associated with metastatic breast cancer.     

Circulating macrophage colony stimulating factor as a marker of tumour progression

Tumour expression of the macrophage colony stimulating factor (CSF-1 or MCSF) has been associated with an adverse prognosis in breast cancer, through an effect on the promotion of metastasis. The aim of the present study was to evaluate the clinical relevance of high circulating CSF-1 levels in patients with newly diagnosed breast tumours and correlate CSF-1 with clinico-pathological parameters. A secondary aim was to also measure CSF-1 in patients with other tumour types and at different stages of disease. Using a commercially available ELISA, pre-treatment plasma levels of CSF-1 were assessed, in 471 consecutive patients diagnosed with breast tumours, in 70 patients with newly diagnosed cancer of the head & neck, in 32 men with prostate cancer metastatic to bone and in 39 women with advanced metastatic breast cancer. Mean CSF-1 levels were significantly higher in patients with locally advanced (p <.015) or metastatic breast tumours (p <.048) and in a group of primary breast cancer patients (n = 26) selected for intensive chemotherapy because of multiple adverse tumour characteristics (p <.0002). Mean CSF-1 was also higher in patients younger than 35 years (p <.02) and in post-menopausal patients (p <.03). There was no significant association with tumour histologic type, grade, or other individual histopathologic parameters. No significant association was found between pre-treatment CSF-1 and overall/relapse free survival. Median CSF-1 levels were dramatically higher in patients with newly diagnosed tumours of the head & neck (604 pg/ml), in men with prostate cancer metastatic to bone (627 pg/ml) and women with advanced metastatic breast cancer (867 pg/ml) than those seen in patients with newly diagnosed breast tumours (334 pg/ml). Our data support the hypothesis that CSF-1 may play a functional role in tumour progression to metastasis as has previously been reported in animal models.     

Correlation of interleukin 6 with interleukin 1alpha in human mammary tumours, but not with oestrogen receptor expression

The authors hypothesized that IL-6 production by breast tumour tissues would correlate with OR-positivity, as only those tumours that contain oestrogen receptors (OR) and use oestrogen as a mitogen would benefit from locally increased oestrogen. IL-6 increases the activity of the 17beta-oxidoreductase, which converts oestrone to oestradiol, a process that may contribute to the increased concentration of oestrogen around breast tumours. IL-1alpha upregulates IL-6 production; therefore, the correlation between IL-1alpha and IL-6 immunoreactivity and OR-positivity in paraffin-embedded human breast tumours was further investigated.The results indicate IL-6 immunoreactivity in 40 of 66 paraffin embedded breast tumour specimens, a finding which did not correlate with the clinical evaluation of oestrogen receptor positivity (P=0.32 by Fisher's exact test). However, there was a correlation between IL-6 and IL-1alpha immunoreactivity (P<0.05). To study an in vitro model for this phenomenon, the IL-6 immunoreactivity in available cell lines was tested. Surprisingly, no production of IL-6 protein or mRNA could be detected in any of the cell lines, and this did not change with IL-1alpha stimulation. Therefore, none of the cell lines apparently reflected the immunological potential observed in the majority of surgical specimens.     

Ultrasound hyperthermia control system for deep-seated tumours: Ex vivo study of excised tumours,modeling of thermal profile and future nanoengineering aspects

Hyperthermia is a technique of raising the temperature locally to treat say tumours. There are several hyperthermia modalities like radio frequency (RF), microwave, and ultrasound. RF and microwave hyperthermia are good for superficial treatment while ultrasound is good for the therapeutic treatment of deep-seated tumours, with the ability of easy focussing. Focussed ultrasound system developed for deep-seated tumours, say in the complex brain tissue, is studied here. Nanotechnological approach is presented here for different control mechanisms for the control of ultrasound intensity, focussing beam, thermal profile of temperature distribution in the tissue and dosage control levels. Ex vivo study of excised tumours, like breast tumours, bone tumours and other such samples, with the present system is also presented. Physical and biological effects on the human health are discussed.     

Ametantrone inhibits prostaglandin--mediated resorption in bone organ culture

Prostaglandins (PG) have been postulated to be involved in both tumor metastases to bone and in tumor-induced bone resorption. The anthracenedione antineoplastic agents ametantrone (HAQ) and mitoxantrone are potent antioxidants and inhibit hydroperoxide-dependent initiation and propagation reactions. Therefore, these compounds may inhibit PG production and could also inhibit tumor metastases and tumor-induced resorption. The ability of HAQ, a prototypic anthracenedione, to inhibit PG synthesis and PG-mediated bone resorption was investigated using neonatal mouse calvaria in organ culture. Epidermal growth factor (EGF) stimulates bone resorption in this tissue by inducing PG synthesis. Consequently, if HAQ inhibits EGF-stimulated PG synthesis, it should also inhibit EGF-stimulated bone resorption. HAQ, at 10 microM, completely abolished EGF-stimulated PG synthesis and calcium release. Moreover, HAQ (1.0-30 microM) inhibition of EGF-stimulated PGE2 synthesis correlated with the inhibition of EGF-stimulated Ca release in a concentration-dependent manner. In contrast to EGF, parathyroid hormone stimulates resorption by a PG-independent pathway. HAQ at 10 microM had no effect on parathyroid hormone stimulated Ca release. These results suggest that HAQ inhibition of bone resorption appears to be primarily mediated by inhibition of PG biosynthesis.     

The primary structure of the core protein of the small, leucine-rich proteoglycan (PG I) from bovine articular cartilage

Two forms of small, interstitial proteoglycans have been isolated from bovine articular cartilage and have different core proteins, based on NH2-terminal analysis and peptide mapping (Choi, H. U., Johnson, T. L., Pal, S., Tang, L-H., Rosenberg, L. C., and Neame, P. J. (1989) J. Biol. Chem. 264, 2876-2884). These proteoglycans have been called PG I and PG II. Since they were first described, they have also been called "biglycan" (PG I), "decorin," and "DS-PG" (PG II). This report describes the primary structure of PG I from bovine articular cartilage. The protein core consists of 331 amino acids with a molecular mass of 37,280 Da. The amino acid sequence shows 55% identity to the cDNA-derived sequence of PG II from bovine bone. There are four discrete domains in the amino acid sequence. Domain 1, at the NH2 terminus (approximately 23 amino acids), contains two sites of attachment of dermatan sulfate, both of which match the consensus sequence of Asp/Glu-X-X-Ser-Gly-hydrophobic. Neither of these sites is substituted to 100% with glycosaminoglycan in native PG I. Domain 2, near the NH2 terminus and containing approximately 28 amino acids, has a cysteine pattern similar to a domain near the COOH terminus of mouse metallothionein and contains at least one disulfide bond (between the first and fourth cysteine residues). The majority of the core protein of PG I (domain 3) is a leucine-rich domain containing ten repeating units (approximately 231 amino acids). Patthy [1987) J. Mol. Biol. 198, 567-577) has shown that for PG II, the majority of domain 3 shows considerable similarity to leucine-rich alpha 2-glycoprotein (LRG) from serum. Domain 2 of PG I or PG II also has an analog in LRG, in that it has two cysteines in a similar place. The major motif in the PG I described here, in PG II and in LRG, is a series of leucine-rich repeats. PG I and PG II both contain 10 leucine-rich repeats which are 14 amino acids long and which are somewhat irregularly spaced, while LRG contains 9 leucine-rich repeats spaced 10 amino acids apart. Other proteins which contain leucine repeats are the platelet glycoprotein Ib, which is involved in platelet adherence to subendothelium (eight repeats in the alpha chain and two in the beta chain), the protein encoded by the Toll gene (involved in lateral and ventral spatial organization in Drosophila) and chaoptin (a protein involved in Drosophila photoreceptor morphogenesis).(ABSTRACT TRUNCATED AT 400 WORDS)     

Ametantrone inhibits prostaglandin — mediated resorption in bone organ culture

Prostaglandins (PG) have been postulated to be involved in both tumor metastases to bone and in tumor-induced bone resorption. The anthracenedione antineoplastic agents ametantrone (HAQ) and mitoxantrone are potent antioxidants and inhibit hydroperoxide-dependent initiation and propagation reactions. Therefore, these compounds may inhibit PG production and could also inhibit tumor metastases and tumor-induced resorption. The ability of HAQ, a prototypic anthracenedione, to inhibit PG synthesis and PG-mediated bone resorption was investigated using neonatal mouse calvaria in organ culture. Epidermal growth factor (EGF) stimulates bone resorption in this tissue by inducing PG synthesis. Consequently, if HAQ inhibits EGF-stimulated PG synthesis, it should also inhibit EGF-stimulated bone resorption. HAQ, at 10 μM, completely abolished EGF-stimulated PG synthesis and calcium release. Moreover, HAQ (1.0–30 μM) inhibition of EGF-stimulated PGE₂ synthesis correlated with the inhibition of EGF-stimulated Ca release in a concentration-dependent manner. In contrast to EGF, parathyroid hormone stimulates resorption by a PG-independent pathway. HAQ at 10 μM had no effect on parathyroid hormone stimulated Ca release. These results suggest that HAQ inhibition of bone resorption appears to be primarily mediated by inhibition of PG biosynthesis.     

Galactolipids and phospholipids of orange peel and juice chromoplasts

Chromoplasts from yellow orange (Citrus sinensis) fruit peel contain monogalactosyl diglycerides (MGDG), digalactosyl diglycerides (DGDG) and phosphatidyl glycerol (PG) in amounts similar to those found in chloroplasts from green fruit peel. Juice chromoplasts contain relatively little MGDG and no DGDG with high levels of phosphatidyl choline and phosphatidyl ethanolamine but no PG.     

Expression of cyclooxygenase-2 in invasive breast carcinomas and its prognostic impact

Representative tumour sections from 468 patients with invasive breast cancer were immunostained for cyclooxygenase-2 (COX-2) and evaluated. The relationships between COX-2 expression, clinical outcome and various clinicopathological variables, including tumour vascularity and disseminated tumour cells (DTC) in the bone marrow were examined. COX-2 expression in invasive breast carcinoma cells was positively associated with oestrogen receptor and/or progesterone receptor positivity (p<0.001). Triple-negative tumours showed no/low COX-2 expression more frequently than other tumour types (p<0.001). Expression of COX-2 was not associated with breast cancer-specific survival (p=0.49, log-rank) or distant disease-free survival (p=0.67, log-rank) for all patients, including lymph node-negative, untreated patients (p>0.14, log-rank). There was also no significant association between COX-2 expression and histological grade, tumour size, nodal status, DTC in bone marrow, p53, HER2, or tumour vascularity. In conclusion, COX-2 expression in this series was associated with the presence of hormone receptors. Low COX-2 expression was observed in triple-negative breast carcinomas.     

Cellular differences in ring glands of flesh fly pupae as a consequence of diapause programming

The ultrastructure of the ring gland (corpus cardiacum (CC), prothoracic gland (PG) and corpus allatum (CA)) was examined in diapausing and nondiapausing flesh fly pupae. The diapause developmental state, which is environmentally regulated and coordinated by the brain-ring gland complex, is associated with differences in the ultrastructure of PG and CA cells but not in the CC. During diapause the PG and CA cells have extensive arrays of rough endoplasmic reticulum and spherical mitochondria. The PG cells also contain lipid droplets surrounded by an electron dense amorphous coat not seen in PG cells from nondiapausing pupae. In nondiapausing pupae, the PG and CA cells contain large amounts of ribosomes throughout the cytoplasm but very little rough endoplasmic reticulum and elongated mitochondria. The fact that ring glands from diapausing pupae readily incorporate (35)S-methioninc indicates that the gland is actively synthesizing proteins, thus the contrasts in ring gland ultrastructure are not due to cellular quiescence during diapause but reflect fundamental cellular and physiological differences between the diapause and nondiapause developmental program.     

Purification and characterization of tomato polygalacturonase converter

Extracts of ripe tomatoes contain two forms of polygalacturonase (PG I and PG II). A heat-stable component that binds PG II to produce PG I has been isolated from tomato fruit. This component has been named polygalacturonase converter (PG converter). The PG converter has been purified by gel filtration, ion-exchange chromatography and chromatofocusing. It appears to be a protein with a relative molecular mass of 102000. It was readily inactivated by papain and pronase. The converter was labile at alkaline conditions, and treatment of PG I at pH 11 released free PG II. A similar factor with a lower molecular mass was extracted from tomato foliage.     

Gamma interferon inhibits basal and interleukin 1-induced prostaglandin production and bone resorption in neonatal mouse calvaria

Production of the osteolytic arachidonic acid metabolites, prostaglandin (PG) E2, PGI2 and PGF2 alpha, by neonatal mouse calvariae was quantitated by gas chromatography/mass spectrometry. Mouse recombinant interleukin 1 (rIL-1) raised medium levels of PGE2 and PGI2 (measured as 6-keto-PGF1 alpha) in the dose range tested (1.0-10.0 U/ml culture medium), while an effect on PGF2 was only observed at 10 U/ml. Bone resorption in response to rIL-1 reached a plateau at 3.0 U/ml. Mouse recombinant gamma-interferon (rIFN-gamma) between 100-500 U/ml suppressed basal PG synthesis and spontaneous resorption of cultured bone. In addition, IFN-gamma at 100 U/ml prevented stimulation of PG synthesis by 3.0 U/ml rIL-1 and thereby reduced the bone resorbing activity of the cytokine by at least 60%. 5 X 10(-7) M indomethacin was equally effective in suppression of PG synthesis and bone resorption. The present study provides evidence that IFN-gamma inhibits PG synthesis and consequently resorption of cultured bone.     

Lipids isolated from bone induce the migration of human breast cancer cells

Bone is the most common site to which breast cancer cells metastasize. We found that osteoblast-like MG63 cells and human bone tissue contain the bile acid salt sodium deoxycholate (DC). MG63 cells take up and accumulate DC from the medium, suggesting that the bone-derived DC originates from serum. DC released from MG63 cells or bone tissue promotes cell survival and induces the migration of metastatic human breast cancer MDA-MB-231 cells. The bile acid receptor farnesoid X receptor (FXR) antagonist Z-guggulsterone prevents the migration of these cells and induces apoptosis. DC increases the gene expression of FXR and induces its translocation to the nucleus of MDA-MB-231 cells. Nuclear translocation of FXR is concurrent with the increase of urokinase-type plasminogen activator (uPA) and the formation of F-actin, two factors critical for the migration of breast cancer cells. Our results suggest a novel mechanism by which DC-induced increase of uPA and binding to the uPA receptor of the same breast cancer cell self-propel its migration and metastasis to the bone.     

Characterization of bone PG II cDNA and its relationship to PG II mRNA from other connective tissues.

Two cDNA clones encoding the small proteoglycan II (PG II) of bone were isolated from a lambda gt11 expression library. These clones expressed recombinant protein which was cross-reactive with polyclonal and monoclonal antisera to PG II molecules from several connective tissues. The longest clone, lambda Pg 20 was studied in detail. The clone was shown to encode PG II by hybrid selected translation and immunoprecipitation. Northern analysis showed two species of the PG II message of approximately 1.4 and 1.8 kb. Substantial amounts of PG II message were found in bone, tendon, articular cartilage, skin, smooth muscle and cornea. Trace amounts of message were also detected in liver and brain. Radiolabeled bovine PG II cDNA hybridized to RNA from several other species including the human, rat and chicken. The level of PG II mRNA in chick embryonic fibroblasts was sensitive to transformation by Rous sarcoma virus.     

Proteoglycans: key partners in bone cell biology

The diversity of bone proteoglycan (PG) structure and localisation (pericellular, extracellular in the organic bone matrix) reflects a broad spectrum of biological functions within a unique tissue. PGs play important roles in organizing the bone extracellular matrix, taking part in the structuring of the tissue itself as active regulators of collagen fibrillogenesis. PGs also display selective patterns of reactivity with several constituents including cytokines and growth factors, such as transforming growth factor-beta or osteoprotegerin thereby modulating their bio-availability and biological activity in the bone tissue. In this review, the complex PG composition in bone will be addressed together with the specific role played by PGs (or their GAGs chains) in bone biology, as regulatory molecules for bone resorption and their involvement in bone tumor development. These roles have been determined after modulation of PG expression or mutations in their corresponding genes, which revealed specific roles for these compounds in bone pathologies (e.g. perlecan or glypican-3 mutations observed respectively in chondrodysplasia or dysmorphic syndrome). Finally, the potential therapeutic interest of PGs is discussed based on recent data, more particularly on bone tumor-associated osteolysis as these molecules are involved both in bone resorption and tumor development.     

Prognostic factors in human primary breast cancer: comparison of c-myc and HER2/neu amplification.

Amplification of oncogenes in primary tumours may have prognostic and/or therapeutic significance for patients with breast cancer. We have studied HER2/neu and c-myc amplification together with steroid receptors in human primary breast tumours and related the outcome with (relapse-free) survival. A strong inverse correlation was found between HER2/neu amplification and the presence of oestrogen and progesterone receptors. Actuarial 5-years survival showed that breast cancer patients with c-myc amplification in their primary tumours experience a shorter relapse-free survival, especially in node-negative and in receptor-positive tumours, whereas HER2/neu amplification may be of prognostic value for overall survival in receptor-negative tumours. Overall, in our hands, c-myc amplification appeared to be a more potent prognosticator than HER2/neu amplification in human primary breast cancer.