The effect of chemical cleansing procedures combined with peracetic acid-ethanol sterilization on biomechanical properties of cortical bone.

Peracetic acid-ethanol sterilization (PES) with a preceding delipidation step is an effective sterilization method for allograft bone, but its influence on biomechanical properties of bone has not been studied. The aim of this study was to evaluate the effects of different incubation times of water, hydrogen peroxide and alcohol cleansing procedures combined with PES on biomechanical properties of freeze-dried cortical bone. These effects were studied by performing three-point bending tests on cortical samples. The lyophilized cortical samples were rehydrated prior to mechanical testing. The bending strength and the absorbed energy of the processed cortical samples were increased slightly but the Young's modulus was decreased compared to unprocessed samples. However, when the residual moisture content of the processed cortical samples was reduced from <5% to 0% all the biomechanical properties studied were significantly decreased. Hexane elution was used to determine the residual fat content of the processed cortical bone. Reducing the incubation time in cleansing had no effect on the residual fat content of the bone samples. Our in vitro study indicates that the cleansing procedure proposed combined with PES affects the biomechanical properties of cortical bone only on a limited scale.

Steroid hormone receptors and coregulators in endocrine-resistant and estrogen-independent breast cancer cells.

Resistance to hormonal therapy is often a problem in the treatment of breast cancer patients. It has been suggested that resistance could be explained by altered nuclear hormone receptor or coregulator levels or inappropriately increased agonist activity of selective estrogen receptor modulator (SERM). To test these hypotheses, we have established novel MCF-7 cell line-derived in vitro models of anti-estrogen- and progestin-resistant and estrogen-independent breast cancer by long-term culture in the presence of toremifene and medroxyprogesterone acetate (MPA) and in the absence of estradiol, respectively. Using cell growth and multiprobe ribonuclease protection assays, the expression of 5 nuclear hormone receptors and 9 coregulators as well as the alterations in the cell proliferation and target gene transcription in response to hormonal treatments were studied. Progesterone receptor (PR) expression was decreased and silencing mediator for retinoid acid and thyroid hormone receptors (SMRT) and amplified in breast cancer-1 (AIB1) expression increased in anti-estrogen-resistant cells. Estrogen caused PR and ERbeta upregulation in all cell lines, but we did not observe increased agonist activity of anti-estrogen measured by regulation of these estrogen target genes. Basal ERalpha levels and estrogenic growth response were decreased and p300/CBP-associated factor (pCAF) and AIB1 upregulated by estrogen in progestin-resistant cells, but coregulator levels were unchanged. Estrogen-independent cells were still estrogen-responsive and PR, nuclear receptor corepressor (N-CoR) and SMRT expression was increased whereas steroid receptor coactivator-1 (SRC-1a) and CBP-related protein p300 (p300) expression decreased. Their growth was inhibited by toremifene, but estradiol was able to abrogate this effect, which might have interesting clinical implications concerning the use of postmenopausal hormone replacement therapy.

G protein-coupled receptor 30 down-regulates cofactor expression and interferes with the transcriptional activity of glucocorticoid.

G protein-coupled receptor 30 (GPR30) has previously been described to be important in steroid-mediated growth and to inhibit cell proliferation. Here we investigated whether the effect of GPR30 on cell growth is dependent on steroid hormone receptors. We stably introduced GPR30 in immortalized normal mammary epithelial (HME) cells using retroviruses for gene delivery. GPR30 inhibited the growth and proliferation of the cells. They expressed glucocorticoid receptor, but not estrogen or progesterone receptor. GPR30 down-regulated the expression of cofactor transcription intermediary factor 2 (TIF2) analyzed using quantitative RT-PCR analysis, and also diminished the expression of TIF2 at protein level analyzed by Western blotting using nuclear extracts from mammary epithelial cells. When HME cells were transiently transfected with the glucocorticoid response element MMTV-luc reporter plasmid, stable expression of GPR30 resulted in the abolition of ligand-induced transactivation of the promoter. In COS cells, transient transfection of GPR30 with glucocorticoid receptor alpha resulted in an abrogation of the MMTV-luc and GRE-luc reporter activities induced by dexamethasone. The results suggest a novel mechanism by which membrane-initiated signaling interferes with steroid signaling.

Expression of nuclear receptors and cofactors in human endometrium and myometrium.

OBJECTIVE: To study the expression of nuclear receptors and cofactors in human endometrium and myometrium in proliferative and secretory phases of the menstrual cycle. METHODS: Multiprobe ribonuclease protection assay and real-time reverse transcriptase polymerase chain reaction were used to quantitate mRNA levels of steroid receptors, vitamin D receptor (VDR), retinoic acid receptors (RAR), and cofactors AIB1 (amplified in breast cancer-1), CBP (cyclic adenosine monophosphate response element binding protein), pCAF (p300/CBP-associated factor), TIF2 (transcription intermediary factor-2), N-CoR (nuclear receptor corepressor), and SMRT (silencing mediator of repressed transcription). Cyclin A expression was analyzed to determine the proliferation status of the tissues. RESULTS: The expression of androgen receptor, estrogen receptors alpha and beta, progesterone receptor, and RARalpha followed cyclin A expression. There was more abundant expression in the proliferative phase endometrium than in the secretory phase endometrium. Glucocorticoid receptor, VDR, RARbeta, and RARgamma were stably expressed during the menstrual cycle in both endometrium and myometrium. Cofactors N-CoR, SMRT, pCAF, CBP, TIF2, AIB1, and p300 mRNAs were expressed in all samples in both endometrium and myometrium. N-CoR, pCAF, AIB1, and p300 appeared not to be regulated when comparing proliferative and secretory phases of the cycle. Individual differences were found in the expression levels of both nuclear receptors and cofactors. CONCLUSION: The menstrual cycle-dependent regulation of nuclear receptor expression was more apparent in the endometrium than in the myometrium, whereas cofactor expression was not cycle dependent. There were individual differences in the expression levels of different receptors and cofactors. In hormonal therapy these differences might result in different responses, depending on the patient as well as the ligand used.

Regulation of nuclear receptor and cofactor expression in breast cancer cell lines.

OBJECTIVE: The aim of this study was to compare the expression profile of nuclear receptors (NRs) and cofactors in different breast cancer cell lines as well as their regulation by estradiol, insulin and progestin R5020. METHODS: Expression of NRs and cofactors were determined from MCF-7, T-47D and ZR-75-1 breast cancer cell lines. Multiprobe ribonuclease protection assay and real-time RT-PCR were used to quantitate mRNA levels of steroid receptors, vitamin D receptors (VDR) and retinoic acid receptors (RAR) and cofactors: amplified in breast cancer-1, cyclic AMP response element binding protein (CBP), p300/CBP-associated factor, p300, nuclear receptor corepressor and silencing mediator of repressed transcription. RESULTS: Basal expression levels of NRs and cofactors varied depending on the cell line. Cell line-specific regulation of androgen receptor, estrogen receptor-alpha (ERalpha), RARalpha, RARgamma and VDR expression was observed after estradiol treatment. Likewise, differences in the regulation of ERalpha, RARalpha and VDR expression after R5020 treatment were observed. We did not observe significant regulation of cofactor expression after estradiol, insulin or progestin treatment in any cell line analyzed. CONCLUSIONS: The results showed that not only is the expression profile of the NRs and cofactors cell line specific but also the regulation of NR expression. Thus the determinants of the ligand action (receptor and cofactor expression) varied considerably among different cell clones of the breast cancer cells. This suggested a gradient of NR-ligand sensitivities in the hormone-dependent breast cancers, which produces an additional challenge in developing novel ligands for hormone replacement therapy and breast cancer treatment.

Expression of progesterone receptor isoforms A and B is differentially regulated by estrogen in different breast cancer cell lines.

Progesterone action in target tissues is mediated through two progesterone receptor (PR) isoforms, PR-A and PR-B, which display different regulatory functions in target cells. Relative expression ratio of these isoforms varies depending on cell and tissue types. Here, we studied the regulation of PR isoform expression by estradiol (E(2)), insulin, IGF-1 and cAMP in different breast cancer cell lines. Although, E(2) induced PR expression in all cell lines studied, the expression ratio of PR-A/PR-B induced by E(2) was dependent on the cell line. The differential regulation of the isoforms was also seen at the mRNA level suggesting that the PR-A and PR-B promoters are differentially regulated by E(2) in different breast cancer cells. Insulin, IGF-1 or cAMP previously reported to induce PR expression however failed to alter the PR expression in our study. This is the first report describing that in different breast cancer cell lines the expression of PR-A and PR-B is regulated by E(2) in a distinct way.

Antiproliferative action of vitamin D.

During the past few years, it has become apparent that vitamin D may play an important role in malignant transformation. Epidemiological studies suggest that low vitamin D serum concentration increases especially the risk of hormone-related cancers. Experimentally, vitamin D suppresses the proliferation of normal and malignant cells and induces differentiation and apoptosis. In the present review we discuss the mechanisms whereby vitamin D regulates cell proliferation and whether it could be used in prevention and treatment of hyperproliferative disorders like cancers.