2‐methoxyestradiol‐mediated anti‐tumor effect increases osteoprotegrin expression in osteosarcoma cells

Osteosarcoma is a bone tumor that frequently develops during adolescence. 2‐Methoxyestradiol (2‐ME), a naturally occurring metabolite of 17β‐estradiol, induces cell cycle arrest and cell death in human osteosarcoma cells. To investigate whether the osteoprotegrin (OPG) protein plays a role in 2‐ME actions, we studied the effect of 2‐ME treatment on OPG gene expression in human osteosarcoma cells. 2‐ME treatment induced OPG gene promoter activity and mRNA levels. Also, Western blot analysis showed that 2‐ME treatment increased OPG protein levels in MG63, KHOS, 143B and LM7 osteosarcoma cells by 3‐, 1.9‐, 2.8‐, and 2.5‐fold, respectively, but did not affect OPG expression in normal bone cells. In addition, increases in OPG protein levels were observed in osteosarcoma cell culture media after 3 days of 2‐ME treatment. The effect of 2‐ME on osteosarcoma cells was ligand‐specific as parent estrogen, 17β‐estradiol and a tumorigenic estrogen metabolite, 16α‐hydroxyestradiol, which do not affect osteosarcoma cell cycle and cell death, had no effect on OPG protein expression. Furthermore, co‐treating osteosarcoma cells with OPG protein did not further enhance 2‐ME‐mediated anti‐tumor effects. OPG‐released in 2‐ME‐treated cultures led to an increase in osteoblastic activity and a decrease in osteoclast number, respectively. These findings suggest that OPG is not directly involved in 2‐ME‐mediated anti‐proliferative effects in osteosarcoma cells, but rather participates in anti‐resorptive functions of 2‐ME in bone tumor environment. J. Cell. Biochem. 109: 950–956, 2010. © 2010 Wiley‐Liss, Inc.     

New embedding medium for sectioning undecalcified bone

Methylmethacrylate (MMA) is the most commonly used embedding medium for sectioning undecalcified bone; however, a number of problems exist with its use in a research laboratory. MMA requires a long infiltration time and temperature control, and it reacts with many polymers. We used Kleer Set resin? as an alternative embedding medium for sectioning undecalcified bone specimens. Fluorochrome labeled bone specimens were sectioned transversely using a ground section technique and longitudinally on a sledge macrotome. The slides were viewed using both transmitted light and epifluorescence microscopy. High quality sections were obtained using Kleer Set resin? for both sectioning techniques. We have shown that this new embedding medium is simpler, safer, quicker to use and does not interfere with visualization of fluorochromes.     

TIEG1-null tenocytes display age-dependent differences in their gene expression, adhesion, spreading and proliferation properties

The remodeling of extracellular matrix is a crucial mechanism in tendon development and the proliferation of fibroblasts is a key factor in this process. The purpose of this study was to further elucidate the role of TIEG1 in mediating important tenocyte properties throughout the aging process. Wildtype and TIEG1 knockout tenocytes adhesion, spreading and proliferation were characterized on different substrates (fibronectin, collagen type I, gelatin and laminin) and the expression levels of various genes known to be involved with tendon development were analyzed by RT-PCR. The experiments revealed age-dependent and substrate-dependent properties for both wildtype and TIEG1 knockout tenocytes. Taken together, our results indicate an important role for TIEG1 in regulating tenocytes adhesion, spreading, and proliferation throughout the aging process. Understanding the basic mechanisms of TIEG1 in tenocytes may provide valuable information for treating multiple tendon disorders.     

Development, characterization, and applications of a novel estrogen receptor beta monoclonal antibody

The role of estrogen receptor alpha (ERα) in breast cancer has been studied extensively, and its protein expression is prognostic and a primary determinant of endocrine sensitivity. However, much less is known about the role of ERβ and its relevance remains unclear due to the publication of conflicting reports. Here, we provide evidence that much of this controversy may be explained by variability in antibody sensitivity and specificity and describe the development, characterization, and potential applications of a novel monoclonal antibody targeting full-length human ERβ and its splice variant forms. Specifically, we demonstrate that a number of commercially available ERβ antibodies are insensitive for ERβ and exhibit significant cross-reaction with ERα. However, our newly developed MC10 ERβ antibody is shown to be highly specific and sensitive for detection of full-length ERβ and its variant forms. Strong and variable staining patterns for endogenous levels of ERβ protein were detected in normal human tissues and breast tumors using the MC10 antibody. Importantly, ERβ was shown to be expressed in a limited cohort of both ERα positive and ERα negative breast tumors. Taken together, these data demonstrate that the use of poorly validated ERβ antibodies is likely to explain much of the controversy in the field with regard to the biological relevance of ERβ in breast cancer. The use of the MC10 antibody, in combination with highly specific antibodies targeting only full-length ERβ, is likely to provide additional discriminatory features in breast cancers that may be useful in predicting response to therapy.     

Blood groups and protein polymorphisms in five goat breeds (Capra hircus)

Data on allele frequencies at six red cell blood group systems and three blood protein polymorphic loci in five goat breeds are reported. Two blood proteins, albumin and carbonic anhy-drase, were not found to be polymorphic. The B blood group system of goats, like its homologue in cattle and sheep, is highly complex. At least 44 B phenogroups (haplotypes) have been distinguished in this study. Based on the variation in allele frequencies between breeds, genetic distances were calculated. The distances estimated by four different methods were in close agreement with data from the history and geographic origins of the breeds examined.     

Effects of immunosuppressants on receptor activator of NF-kappaB ligand and osteoprotegerin production by human osteoblastic and coronary artery smooth muscle cells

Osteoporosis and vasculopathy are common after organ transplantation and have been largely attributed to the use of immunosuppressants. Osteoprotegerin (OPG) is produced by osteoblastic and arterial cells, and inhibits osteoclast functions by neutralizing receptor activator of NF-kappaB ligand (RANKL). Because OPG-deficient mice develop osteoporosis and arterial calcification, we assessed the effects of immunosuppressants on OPG and RANKL expression by human osteoblastic and coronary artery smooth muscle cells (CASMC). Cyclosporine A, rapamycin, and FK-506 decreased OPG mRNA and protein levels in undifferentiated marrow stromal cells (by 63, 44, and 68%, respectively, P < 0.001). All three immunosuppressants increased RANKL mRNA levels in these cells by 60 to 210%. In contrast to these effects on marrow stromal cells, rapamycin, which may be relatively bone-sparing, increased OPG mRNA and protein production (by 120%, P < 0.001) in mature osteoblastic cells. Cyclosporine A also decreased OPG mRNA and protein production (by 52%, P < 0.001) of CASMC. In conclusion, immunosuppressants decrease OPG mRNA and protein production and increase RANKL gene expression by marrow stromal cells, and cyclosporine suppresses OPG production in CASMC. These studies thus provide a potential mechanism for immunosuppressant-induced bone loss, and the propensity of cyclosporine A to cause vascular disease.     

High content image analysis for human H4 neuroglioma cells exposed to CuO nanoparticles

Background  

High content screening (HCS)-based image analysis is becoming an important and widely used research tool. Capitalizing this technology, ample cellular information can be extracted from the high content cellular images. In this study, an automated, reliable and quantitative cellular image analysis system developed in house has been employed to quantify the toxic responses of human H4 neuroglioma cells exposed to metal oxide nanoparticles. This system has been proved to be an essential tool in our study.