Abstract: Fibroblast growth factor 8 (FGF-8) is expressed at an increased level in a high proportion of prostate cancers and it is associated with a poor prognosis of the disease. Our aim was to study the effects of FGF-8b on proliferation of PC-3 prostate cancer cells and growth of PC-3 tumors, and to identify FGF-8b-associated molecular targets. Expression of ectopic FGF-8b in PC-3 cells caused a 1.5-fold increase in cell proliferation in vitro and a four- to fivefold increase in the size of subcutaneous and orthotopic prostate tumors in nude mice. Tumors expressing FGF-8b showed a characteristic morphology with a very rich network of capillaries. This was associated with increased spread of the cancer cells to the lungs as measured by RT-qPCR of FGF-8b mRNA. Microarray analyses revealed significantly altered, up- and downregulated, genes in PC-3 cell cultures (169 genes) and in orthotopic PC-3 tumors (61 genes). IPA network analysis of the upregulated genes showed the strongest association with development, cell proliferation (CRIP1, SHC1), angiogenesis (CCL2, DDAH2), bone metastasis (SPP1), cell-to-cell signaling and energy production, and the downregulated genes associated with differentiation (DKK-1, VDR) and cell death (CYCS). The changes in gene expression were confirmed by RT-qPCR. In conclusion, our results demonstrate that FGF-8b increases the growth and angiogenesis of orthotopic prostate tumors. The associated gene expression signature suggests potential mediators for FGF-8b actions on prostate cancer progression and metastasis. J. Cell. Biochem. (c) 2009 Wiley-Liss, Inc.

Abstract: BACKGROUND: Metastatic prostate cancer is associated with a high morbidity and mortality but the spreading mechanisms are still poorly understood. The aminobisphosphonate alendronate, used to reduce bone loss, has also been shown to inhibit the invasion and migration of prostate cancer cells in vitro. We used a modified orthotopic PC-3 nude mouse tumor model of human prostate cancer to study whether alendronate affects prostate tumor growth and metastasis. METHODS: PC-3 cells (5 x 10(5)) were implanted in the prostates of nude mice and the mice were treated with alendronate (0.5 mg/kg/day in PBS, s.c.) or vehicle for 4 weeks. After sacrifice, the sizes of tumor-bearing prostates were measured and the tumors and prostate-draining regional iliac and sacral lymph nodes were excised for studies on markers of proliferation, apoptosis, angiogenesis and lymphangiogenesis, using histomorphometry and immunohistochemistry. RESULTS: Tumor occurrence in the prostate was 73% in the alendronate-treated group and 81% in the control group. Mean tumor size (218 mm3, range: 96-485 mm3, n = 11) in the alendronate-treated mice was 41% of that in the control mice (513 mm3, range: 209-1350 mm3, n = 13) (p < 0.05). In the iliac and sacral lymph nodes of alendronate-treated mice, the proportion of metastatic area was only about 10% of that in control mice (p < 0.001). Immunohistochemical staining of tumor sections showed that alendronate treatment caused a marked decrease in the number of CD34-positive endothelial cells in tumors (p < 0.001) and an increase in that of ISEL positive apoptotic cells in tumors as well as in lymph node metastases (p < 0.05) compared with those in the vehicle-treated mice. The density of m-LYVE-1-stained lymphatic capillaries was not changed. CONCLUSION: Our results demonstrate that alendronate treatment opposes growth of orthotopic PC-3 tumors and decreases tumor metastasis to prostate-draining lymph nodes. This effect could be at least partly explained by decreased angiogenesis and increased apoptosis. The results suggest that bisphosphonates have anti-tumoral and anti-invasive effects on primary prostate cancer.

Abstract: BACKGROUND AND PURPOSE: Several selective oestrogen receptor modulators (SERMs) with oestrogen agonist effects in bone cells and without increased risk of breast and endometrial cancer have been developed. Here, we have investigated the effects of different types of SERMs on osteoclast differentiation, bone resorption and apoptosis in vitro. EXPERIMENTAL APPROACH: Human peripheral blood-derived CD14+ monocytes were cultured on bovine bone slices in the presence of RANKL, M-CSF, TNF-alpha and dexamethasone for seven days. Also, CD14+ monocytes were co-cultured either with human SaOS-2 or MG-63 osteosarcoma cells, in the presence of parathyroid hormone. Osteoclast cultures were treated with different SERMs. TRACP+ multinucleated cells and C-terminal telopeptide of type I collagen were used as markers for osteoclast formation and bone resorption, respectively. KEY RESULTS: In CD14+ monocyte cultures, tamoxifen directly inhibited human osteoclast formation and bone resorption, while raloxifene and ospemifene had no inhibitory effect. In the co-cultures either with SaOS-2 or MG-63 cells, ospemifene and raloxifene as well as tamoxifen inhibited osteoclast formation in a concentration-dependent manner. The inhibitory effect was associated with an increased production of osteoprotegerin. The anti-oestrogen ICI 182 780 completely reversed the effects of these SERMs. CONCLUSION AND IMPLICATIONS: Tamoxifen had an oestrogen receptor dependent, direct, inhibitory effect on human osteoclast differentiation and bone resorption, whereas ospemifene and raloxifene required osteoblastic cells to achieve a similar inhibition. The effects of ospemifene and raloxifene were mediated by oestrogen receptors by a mechanism involving paracrine induction of osteoprotegerin in cultures with osteoblast derived osteosarcoma cells.

Abstract: A method is introduced for quantitative detection of cell surface protein expression. The method is based on immunocytochemistry, the use of long decay time europium(III) chelate and platinum(II) porphyrin labels, and detection of photoluminescence emission from adhered cells by time-resolved fluorimetry. After immunocytochemistry, the assay wells are evaporated to dryness and measured in the dry state. This protocol allows repeated and postponed analysis and microscopy imaging. In order to investigate the performance of the method, we chose expression of intercellular adhesion molecule-1 (ICAM-1) of endothelial cell line EAhy926 as a research target. The expression of ICAM-1 on the cells was enhanced by introduction of a cytokine, tumour necrosis factor-alpha (TNFalpha). The method gave signal:background ratios (S:B) of 20 and 9 for europium and platinum labels, respectively, whereas prompt fluorescent FITC label gave a S:B of 3. Screening window coefficients (=Z'-factor) were >0.5 for all the three labels, thus indicating a score for an excellent screening assay. In conclusion, the method appears to be an appropriate choice for protein expression analysis, both in high-throughput screening applications, and for detailed sample investigation by fluorescent microscopy imaging.

Abstract: Hormonal cancers such as breast and prostate cancer arise from steroid hormone-regulated tissues. In addition to breast and prostate cancer hormonal regulation has also a role in endometrial, ovarian, testis and thyroid carcinomas. The effects of estrogens, androgens and progestagens on tumor growth are largely mediated by paracrine and autocrine target molecules which include growth factors and growth factor receptors. During cancer progression the hormonal growth regulation is often lost or overcome by an inappropriate activation of growth factor signaling cascades. One of the growth factors which have been associated with the regulation of growth and progression of hormonal cancer is fibroblast growth factor 8 (FGF8) which has also been recognized as an oncogene. FGF8 is widely expressed during embryonic development. It has been shown to mediate embryonic epithelial-mesenchymal transition and to have a crucial role in gastrulation and early organization and differentiation of midbrain/hindbrain, pharyngeal, cardiac, urogenital and limb structures. During adulthood FGF8 expression is much more restricted but in hormonal cancers it becomes frequently activated. High level of FGF8 expression in tumors is associated with a poor prognosis at least in prostate cancer. In experimental models FGF8 induces and facilitates prostate tumorigenesis and increases growth and angiogenesis of tumors. Several lines of evidence for autocrine and paracrine loops in the growth regulation of breast, prostate and ovarian cancer by FGF8 have been suggested.

Abstract: Several members of the fibroblast growth factor (FGF) family have an important role in the development of skeletal tissues. FGF-8 is widely expressed in the developing skeleton, but its function there has remained unknown. We asked in this study whether FGF-8 could have a role in the differentiation of mesenchymal stem cells to an osteoblastic lineage. Addition of FGF-8 to mouse bone marrow cultures effectively increased initial cell proliferation as well as subsequent osteoblast-specific alkaline phosphatase production, bone nodule formation, and calcium accumulation if it was added to the cultures at an early stage of osteoblastic differentiation. Exogenous FGF-8 also stimulated the proliferation of MG63 osteosarcoma cells, which was blocked by a neutralizing antibody to FGF-8b. In addition, the heparin-binding growth factor fraction of Shionogi 115 (S115) mouse breast cancer cells, which express and secrete FGF-8 at a very high level, had an effect in bone marrow cultures similar to that of exogenous FGF-8. Interestingly, experimental nude mouse tumors of S115 cells present ectopic bone and cartilage formation as demonstrated by typical histology and expression of markers specific for cartilage (type II and IX collagen) and bone (osteocalcin). These results demonstrate that FGF-8 effectively predetermines bone marrow cells to differentiate to osteoblasts and increases bone formation in vitro. It is possible that FGF-8 also stimulates bone formation in vivo. The results suggest that FGF-8, which is expressed by a great proportion of malignant breast and prostate tumors, may, among other factors, also be involved in the formation of osteosclerotic bone metastases.

Abstract: In the search for androgen target genes responsible for malignant growth in S115 mouse mammary tumor cells we found that thrombospondin 1 (TSP1) expression was strongly downregulated by testosterone (Te). Experiments with cycloheximide suggested that Te repression of TSP1 was dependent on de novo protein synthesis. TSP1 repression by Te was preceded by the induction of fibroblast growth factor 8 (FGF8) expression. FGF8 has previously been shown to mediate androgen effects on proliferation of S115 cells by autocrine/paracrine mechanisms. It has also been shown to increase breast cancer cell growth as tumors in nude mice and to stimulate tumor angiogenesis. We studied here the possibility that FGF8 belonged to the Te-induced de novo synthesized proteins that mediate the effect of Te on TSP1 expression in these cells. We found that addition of FGF8b to in vitro cultures or ectopic expression of FGF8b in S115 cells repressed TSP1 expression at mRNA and protein levels even in the absence of Te. FGF2, another angiogenic member of FGF family, also downregulated TSP1 mRNA level in the in vitro cultures of S115 cells. The antisense oligonucleotides for FGF8 did not, however, prevent Te-repression of TSP1 mRNA expression and a neutralizing anti-FGF8b antibody only partially opposed Te induced downregulation of TSP1. These results suggest that both androgen and FGF8 inhibit TSP1 expression independently. They also suggest that opposite to many other androgen-induced responses in S115 cells, the effect of Te on the expression TSP1 is not mediated by FGF8.

Abstract: Postmenopausal decline of estrogen production is associated with development of several degenerative disorders such as osteoporosis, neuroinflammatory diseases and vascular wall degeneration. These are associated with the activation of the cells of the monocyte-macrophage system in a context-dependent manner. Estrogen regulates differentiation, maturation and function of many cell types in this system directly or indirectly via other cells by autocrine/paracrine mechanisms. Estrogen effects on the monocyte-macrophage system are primarily repressive. Most of these effects are mediated by repression of expression of genes for cytokines or modulation of other inflammatory mediators by the estrogen receptor (ER)-dependent or nongenomic pathways. The ER-dependent mechanisms mostly involve modulation of the nuclear factor kappa B (NF-kappaB) pathway for transcriptional regulation of cytokine or other mediator genes. In the context of hormone-regulated cancer, estrogen can influence production of cytokines or other inflammatory mediators by both tumor cells and tumor-invading macrophages. The interactions of breast and prostate cancer cells with tumor-associated macrophages (TAMs) may play an important role in tumor progression and even in the development of resistance to hormonal treatment. Regulation of the monocyte-macrophage system by estrogen and cross-talk between the ER and cytokine-mediated pathways provides multiple novel targets for development of selective ER modulator (SERM) molecules for prevention and treatment of postmenopausal degenerative and neoplastic diseases.

Abstract: A study on cytotoxic effect of acetone extract of "Triphala" whose antimutagenicity has already been tested. The in vitro antimutagenic activity of Triphala--an Indian herbal drug. Food Chemistry and Toxicology 40, 47-54) was extended to test its cytotoxic effects on cancer cell-lines using Shionogi 115 (S115) and MCF-7 breast cancer cells and PC-3 and DU-145 prostate cancer cells as models. The results revealed that acetone extract of "Triphala" showed a significant cytotoxic effect on these cancer cell-lines and the effect was similar on all cancer cell lines used in this study. The major phenolic compounds in the most potent acetone extracts were isolated and purified. Structural analysis was conducted using spectroscopic techniques including mass spectroscopy, nuclear magnetic resonance (NMR) and infrared (IR) which showed gallic acid as the major component. The suppression of the growth of cancer cells in cytotoxic assays may be due to the gallic acid-a major polyphenol observed in "Triphala".

Abstract: Glucocorticoid-induced osteoporosis may be at least in part due to the increased apoptosis of osteocytes. To study the role of osteocyte apoptosis in glucocorticoid-induced osteoporosis, we isolated primary osteocytes from murine calvaria for the analysis of the effects of dexamethasone in in vitro culture. The cells were identified by morphology, cytochemical staining, immunocytochemical staining and mRNA expression of phosphate-regulating gene with homology to endopeptidases on the X chromosome (PHEX) and sclerosteosis/van Buchem disease gene (SOST). We found that dexamethasone induced osteocyte apoptosis in a dose-dependent manner. A glucocorticoid receptor antagonist, mifepristone (RU486), suppressed dexamethasone-induced osteocyte apoptosis, suggesting that it was mediated by glucocorticoid receptor. Immunocytochemical stainings showed that glucocorticoid receptors are present in primary osteocytes, and they were translocated to nuclei after the exposure to dexamethasone. Addition of estrogen prevented glucocorticoid receptor translocation into nuclei. Corresponding antiapoptotic effects in primary osteocytes were also seen after the pretreatment of primary osteocytes with a picomolar concentration of estrogen. The pure antiestrogen ICI 182,780 inhibited estrogen effect on apoptosis induced by dexamethasone. These data suggest that glucocorticoid receptors play an important role in glucocorticoid-induced osteocyte apoptosis. Most importantly, estrogen has a protective effect against osteocyte apoptosis. To conclude, the mechanism of glucocorticoid-induced osteoporosis may be due to the apoptosis of osteocytes, which can be opposed by estrogen.

Abstract: Using human peripheral blood CD14(+) osteoclast precursors, we show that testosterone directly inhibits osteoclast formation and bone resorption at physiological concentrations. Instead, estrogen has no direct effects, whereas its action seems to be mediated through osteoblasts by producing osteoprotegerin. Both estrogen and testosterone acts through their cognate receptors. INTRODUCTION: Estrogen (E2) deficiency is associated with both the development of postmenopausal and senile form of osteoporosis in elderly women. Testosterone (Te) deficiency, on the other hand, may cause osteoporosis in men. In both sexes, osteoporosis is associated with disturbed bone turnover, including increased bone resorption caused by enhanced osteoclast formation and increased osteoclast activity. However, the mechanisms by which E2 or Te act on bone are not fully understood, and one of the central questions is whether these hormones act directly on osteoclast precursors or whether their action is mediated through osteoblastic cells. MATERIALS AND METHODS: We cultured human peripheral blood CD14(+) osteoclast precursors in the presence of RANKL, macrophage-colony stimulating factor (M-CSF), TNF-alpha, and dexamethasone to induce them to differentiate into osteoclasts. To study the possible osteoblast-mediated effects, osteoclast precursors were also co-cultured either with human MG-63 or SaOS-2 osteoblast-derived osteosarcoma cells. These cultures were treated with 10(-8)-10(-12) M of E2 or Te for 7 days. RESULTS: E2 did not have any direct effect on osteoclast formation, whereas testosterone inhibited osteoclast formation and bone resorption in a dose-dependent manner. In co-cultures, where MG-63 or SaOS-2 cells were present, E2 and Te inhibited osteoclast formation in a dose-dependent manner. At the same time, E2 and Te treatment in MG-63 or SaOS-2 cell-containing cultures stimulated significantly the formation of osteoprotegerin (OPG) compared with untreated cultures measured by ELISA assay from the culture medium. The effects of E2 and Te on osteoclast formation and bone resorption were completely antagonized by an E2 receptor (ER) antagonist, ICI 182,780, and an androgen receptor (AR) antagonist, flutamide, suggesting ER- and AR-mediated mechanisms, respectively, in these cultures. CONCLUSIONS: Te is likely to have direct and indirect inhibitory effects on human osteoclast formation and bone resorption, whereas the effect of E2 on osteoclast precursors and osteoclasts seems to be mediated by osteoblastic cells. Inhibitory effect of E2 is associated with the stimulated secretion of OPG by osteoblast-derived osteosarcoma cells. Mechanism of action of E2 and Te is mediated by ER and AR, respectively.

Abstract: Fluorescence polarization is one of the most commonly used homogeneous assay principles in drug discovery for screening of potential lead compounds. In this article, the fluorescence polarization technique is combined with 2-photon excitation of fluorescence. Theoretically, the use of 2-photon excitation of fluorescence increases the volumetric sensitivity and polarization contrast of fluorescence polarization assays. The work in this report demonstrates these predictions for an estrogen receptor ligand binding assay.

Abstract: Estrogens have previously been extensively used in prostate cancer treatment. Serious side effects, primarily in cardiovascular system have, however, limited their use. The therapeutic effect of estrogen in preventing prostate cancer growth was mainly obtained indirectly by feedback inhibition of the hypothalamic release of LRH leading to lowered serum androgen levels and castration like effects. Prostate tissue is also most probably a target for direct regulation by estrogens. Prostate contains estrogen receptor alpha (ERalpha) and beta (ERbeta), which are localized characteristically in stroma and epithelium, respectively. The physiological function of these receptors is not known but there is evidence of the role of estrogens in prostatic carcinogenesis. Developing prostate seems particularly sensitive to increased level of endogenous and/or exogenous estrogens. Perinatal or neonatal exposure of rats and mice to estrogens leads to "imprinting" of prostate associated with increased proliferation, inflammation and dysplastic epithelial changes later in life. Prolonged treatment of adult rodents with estrogens along with androgens also leads to epithelial metaplasia, PIN-like lesions and even adenocarcinoma of prostate speaking for the role of estrogen in prostate cancer development. Recent results concerning antiestrogen inhibition of prostate cancer development beyond PIN-type lesions in transgenic mouse models further suggests a role for estrogens in prostate cancer progression. These results also suggest that direct inhibition of estrogen action at the level of prostate tissue may provide an important novel principle of development of prostate cancer therapies.

Abstract: To investigate the differential short-term effects of selective estrogen receptor (ER) modulators (SERMs) on uterus, we treated adult ovariectomized rats with a novel SERM, ospemifene (Osp), two previously established SERMs (tamoxifen and raloxifene (Ral)) and estradiol. The expression of two estrogen-regulated early response genes c-fos and vascular endothelial growth factor (VEGF), and DNA synthesis were analysed at 1-24 h after treatment of ovariectomized rats. Induction of c-fos mRNA by each of the SERMs showed a biphasic pattern with peaks at 3 and 20 h, respectively. The maximum level of VEGF mRNA was observed at 1 h after raloxifene and 6 h after tamoxifen or ospemifene treatment. Maximum levels of the c-fos and VEGF mRNA after raloxifene treatment were higher than those seen after treatments with E2 or a corresponding dose of tamoxifen or ospemifene. DNA synthesis was significantly increased by ospemifene, tamoxifen and raloxifene both in luminal and glandular epithelium. The stimulation was transient, peaking at 16 h. In comparison, the maximum level observed at 16 h after E2 treatment sustained at least until 24 h. DNA synthesis in stromal cells was increased by the SERMs but not by E2 at 24 h. When treated together with E2, the SERMs were able to antagonise E2-stimulated DNA synthesis at 16 h. Our results demonstrate that the initial response of uterus to ospemifene, raloxifene and tamoxifen includes activation of early response genes and even transient stimulation of DNA synthesis in spite of their different long-term effects. However, the early stimulatory events may be mediated by different mechanisms leading to diverging pathways in various tissue compartments and development of differential SERM-specific long-term responses of uterus.

Abstract: Bone remodeling involves old bone resorption by osteoclasts and new bone formation by osteoblasts. However, the precise cellular mechanisms underlying these consecutive events remain obscure. To address this question in vitro, we have established a cell culture model in which the resorption lacunae are first created by osteoclasts and osteoblast-like cells accomplish the subsequent bone formation. We isolated osteoclasts from rat bone marrow and cultured them on bovine bone slices for 48 hours to create resorption lacunae. After removing osteoclasts, confluent differentiated primary osteoblast cultures were trypsinized and the cells were replaced on the resorbed bone slices for up to 14 days. The cultures were then examined by confocal microscopy, field emission scanning electron microscopy (FESEM), and transmission electron microscopy (TEM). Our data suggest that after osteoclastic bone resorption, osteoblast-like cells, not macrophages, remove the remaining organic matrix in the lacuna. After cleaning the lacuna, osteoblast-like cells deposit new collagen fibrils at the bottom of the lacuna and calcify the newly formed matrix only, as visualized by labeled tetracycline accumulation merely in the lacuna during the osteoblast culture. Furthermore, an electron-dense layer rich in osteopontin separates the old and new matrices suggesting formation of the cement line. Since the morphology of the newly formed matrix is similar to the natural bone with respect to the cement line and osteoid formation as well as matrix mineralization, the present method provides for the first time a powerful in vitro method to study the cellular mechanisms leading to bone remodeling also in vivo.

Abstract: Selective estrogen receptor modulators (SERMs), tamoxifen (Tam) and toremifene (Tor), are widely used in the treatment of breast cancer. In addition, they have been demonstrated to prevent estrogen deficiency-induced bone loss in postmenopausal women. These effects are thought to be caused by the interaction of the SERMs with the estrogen receptor, although SERMs have also been shown to conduct non-receptor-mediated effects such as rapid changes in membrane functions. We compared the effects of Tam, Tor, and 17beta-estradiol (E2) on the viability of rat osteoclasts and osteoblasts. Both Tam and Tor were found to cause osteoclast apoptosis in in vitro cultures, which was reversed by E2. In addition, at higher concentration (10 microM), both SERMs had an estrogen receptor-independent effect, which involved interaction with the plasma membrane as demonstrated with UMR-108 osteosarcoma cells by Tam and Tor, but not E2. A leak of protons leading to changes in intracellular pH was shown both in medullary bone derived membrane vesicles and in intact cells. These effects were followed by a rapid loss of cell viability and subsequent cell lysis. Our results show that both Tam and Tor have an ionophoric effect on the plasma membranes of bone cells and that these SERMs differed in this ability: Tor induced rapid membrane depolarization only in the presence of high concentration of potassium. These non-receptor-mediated effects may be involved in therapeutic responses and explain some clinical side effects associated with the treatment of patients with these SERMs.

Abstract: We investigated the effect of water and acetone extract of Juglans regia L. to evaluate its antimutagenic and antiproliferative activities. The antimutagenic study using TA98 and TA100 tester strains of Salmonella revealed the water and acetone extracts to be more effective than the benzene and chloroform extracts in inhibiting the revertants induced by 2-aminoflourene (2AF) in TA100 tester strains. The most effective extracts in the Ames assay were further evaluated using the Lucifer luciferase assay and in time course studies for antiproliferative activities using the Hoechst staining to observe apoptotic cell deaths. The acetone extract showed a correlation of antimutagenic activities in the Ames assay with its antiproliferative effect in different cell lines, while the water extract exerted its effect distinctly in each cell line. Further studies are still needed to evaluate the cytotoxicity in experiments carried out in vivo.

Abstract: Using long-term organ cultures of rat prostate tissue explants, we previously demonstrated that PRL both stimulates proliferation and acts as an androgen-independent suppressor of apoptosis in prostate epithelial cells, leading to epithelial hyperplasia. In this work we delineate intracellular signaling molecules activated by PRL in prostate tissue to identify candidate signaling proteins that are responsible for maintaining survival and proliferation of prostate epithelium in androgen-deprived growth environment. We now show that signal transducer and activator of transcription-5a (Stat5a) and Stat5b become tyrosine phosphorylated in response to PRL stimulation in rat prostate using prostate organ culture as an experimental model. Stat5 was translocated to the nuclei of epithelial cells of prostate tissue as demonstrated by immunohistochemistry. Furthermore, EMSA showed PRL-inducible binding of Stat5a homodimers and Stat5a/5b heterodimers to the PRL response element of the beta-casein gene promoter. Signaling molecules Stat3, Stat1, MAPK, or protein kinase B, which can be activated by PRL in other target cells, were not activated by PRL in prostate tissue. Furthermore, we show that Stat5a and Stat5b are continuously phosphorylated in rat prostate in vivo, although they are expressed to varying degree in separate lobes of rat prostate. Collectively, our results suggest that PRL signaling in rat prostate tissue is primarily transduced via Stat5a and Stat5b. The Stat5 pathway represents one candidate signaling mechanism, used by PRL and possibly other growth factors and cytokines, that supports the viability of prostate epithelial cells during long-term androgen deprivation.

Abstract:

Abstract: The spread of cancer cells to regional lymph nodes through the lymphatic system is the first step in the dissemination of breast cancer. In several human cancers including those of the breast and prostate, the expression of vascular endothelial growth factor C (VEGF-C) is associated with lymph node metastasis. Our study was undertaken to evaluate the effect of VEGF-C on metastasis of poorly invasive, estrogen dependent human MCF-7 breast cancer cells. MCF-7 breast cancer cells transfected with VEGF-C (MCF-7-VEGF-C) were grown as tumors in the mammary fat pads of nude mice implanted with subcutaneous estrogen pellets. Tumor lymphangiogenesis and lymph node metastasis were studied immunohistochemically using antibodies against lymphatic vessel hyaluronan receptor -1 (LYVE-1), VEGF receptor-3 (VEGFR-3), PECAM-1, pan-cytokeratin and estrogen dependent pS2 protein. Overexpression of VEGF-C in transfected MCF-7 cells stimulated in vivo tumor growth in xenotransplanted mice without affecting estrogen responsiveness. The resulting tumors metastasized to the regional lymph nodes in 75% (in 6 mice out of 8, Experiment I) and in 62% (in 5 mice out of 8, Experiment II) of mice bearing orthotopic tumors formed by MCF-7-VEGF-C cells whereas no metastases were observed in mice bearing tumors of control vector-transfected MCF-7 cells (MCF-7-Mock). The density of intratumoral and peritumoral lymphatic vessels was increased in tumors derived from MCF-7-VEGF-C cells but not MCF-7-Mock cells. Taken together, our results show that VEGF-C overexpression stimulates tumor lymphangiogenesis and induces normally poorly metastatic estrogen-dependent MCF-7 tumors to disseminate to local lymph nodes. These data suggest that VEGF-C has an important role in lymph node metastasis of breast cancer even at its hormone-dependent early stage.

Abstract: Breast and prostate cancer preferentially metastasize in the skeleton, inducing locally increased bone resorption by osteoclasts. Bisphosphonates (BPs), potent inhibitors of osteoclasts and bone resorption, are able to reduce metastatic bone lesions, but the metastasis-related cellular target molecules for BPs have not yet been identified. In osteoclasts, nitrogen-containing BPs inhibit the function of the mevalonate pathway, impairing the prenylation and activation of small GTPases. In addition, direct effects of BPs on cancer cells have been suggested. In the present study, the effects of two clinically used BPs, the amino-BP alendronate and clodronate, on adhesion, invasion, and migration of human PC-3 prostate cancer cells were examined in vitro. We also studied the possible role of the mevalonate pathway in invasion and migration of PC-3 cells using the beta-hydroxy-beta-methylglutaryl-CoA reductase inhibitor mevastatin and the mevalonate pathway intermediates mevalonate (mevalonic acid lactone), geranylgeraniol, and trans-trans-farnesol. The results demonstrate that alendronate pretreatment very effectively inhibited in vitro invasion of prostate cancer cells in a dose-dependent manner, with an IC50 as low as approximately 1 pM. The inhibition was similar to that of mevastatin. Clodronate also inhibited invasion, but the IC50 was 0.1 microM. Importantly, geranylgeraniol and trans-trans-farnesol reversed the inhibitory effect of alendronate and mevastatin but not the clodronate-induced inhibition of invasion. Alendronate pretreatment also inhibited migration, which was partially reversed by geranylgeraniol and trans-trans-farnesol. Adhesion of PC-3 cells to various matrices was reduced, and their F-actin organization was changed. Alendronate pretreatment also inhibited invasion of human Du-145 prostate and MDA-MB-231 breast cancer cells. As a conclusion, the results demonstrate that the mevalonate pathway leading to protein prenylation is important for cancer cell invasion and migration in vitro. They further suggest that interference with this pathway is involved in inhibition of invasion and migration of prostate cancer cells by the amino-BP alendronate but that the mechanism of clodronate inhibition is different. It is possible that BPs have therapeutic potential in preventing the spread of prostate cancer.

Abstract: SUMMARY: Fibroblast growth factor 8 (FGF-8) is implicated in growth of prostate cancer. Alternative splicing of the human FGF-8 gene potentially allows coding for four protein isoforms (a, b, e, and f). These isoforms differ in their binding to FGF receptors (FGFR) and in their mitogenic and transforming capacity in transfection assays. Here, we used RT-PCR and immunohistochemistry to study the expression of FGF-8 and FGFR isoforms in human prostate cancer (n = 31). Nonmalignant prostate specimens from cystoprostatectomies (n = 24) were examined as controls. Most prostate cancer samples and some control prostates also contained prostatic intraepithelial neoplasia (PIN) lesions. FGF-8a and e were expressed at significantly higher frequencies in prostate cancer (FGF-8a, 55%; FGF-8e, 45%) than in control samples (FGF-8a, 17%, p = 0.0052; FGF-8e, 8%, p = 0.0031). On the contrary, FGF-8b was found at an equal frequency in prostate cancer (55%) and in control prostates (50%). Furthermore, a combination of two or three FGF-8 isoforms (a, b, and/or e) was also expressed at a higher frequency in prostate cancer than in control samples (45% and 8%, respectively, p = 0.0031). Immunohistochemistry with an antibody recognizing all FGF-8 isoforms was more strongly immunoreactive in prostate cancer cells and PIN lesions than in normal-type epithelium. The receptor splicing variants FGFR1IIIc and FGFR2IIIc, which are activated by FGF-8, were found both in prostate cancer and control samples. Interestingly, immunoreactivity for FGFR1 and FGFR2 was much stronger in prostate cancer cells and PIN than in normal epithelium. These results demonstrate, for the first time, that FGF-8 isoforms and their receptors FGFR1IIIc and FGFR2IIIc are expressed at an increased level not only in prostate cancer but also in premalignant PIN lesions. These data suggest that FGF-8 may have an important autocrine role in the development of human prostate cancer. In addition to FGF-8b, the FGF-8 isoforms a and e may be involved in this process.

Abstract: Fibroblast growth factor 8 (FGF-8) is a secreted heparin-binding protein, which has mitogenic and transforming activity. Increased expression of FGF-8 has been found in human breast cancer, and it has a potential autocrine role in its progression. Human FGF-8 is alternatively spliced to generate four protein isoforms (a, b, e, and f). Isoform b has been shown to be the most transforming. In this work, we studied the role of FGF-8b in the growth (in vitro and in vivo) of MCF-7 human breast cancer cells, which proliferate in an estrogen-dependent manner. Constitutive overexpression of FGF-8b in MCF-7 cells down-regulated FGF-8b-binding receptors FGF receptor (FGFR) 1IIIc, FGFR2IIIc, and FGFR4 found to be expressed in these cells. FGF-8b overexpression led to an increase in the anchorage-independent proliferation rate in suspension culture and colony formation in soft agar, when MCF-7 cells were cultured with or without estradiol. FGF-8b also provided an additional growth advantage for cells stimulated with estradiol. In addition, FGF-8b-transfected cells invaded more actively through Matrigel than did control cells. This was possibly due to the increased secretion of matrix metalloproteinase 9. In vivo, FGF-8b-transfected MCF-7 cells formed faster growing tumors than vector-only-transfected cells when xenografted into nude mice. The tumors formed by FGF-8b-transfected cells were more vascular than the tumors formed by vector-only-transfected cells. In conclusion, FGF-8b expression confers a growth advantage to MCF-7 breast carcinoma cells, both in vitro and in vivo. In addition to stimulation of proliferation, this growth advantage probably arises from increased invasion and tumor vascularization induced by FGF-8b. The results suggest that FGF-8b signaling may be an important factor in the regulation of tumorigenesis and progression of human breast cancer.

Abstract: The Shionogi 115 (S115) mouse mammary tumor cells express the MMTV-specific 1.7 kb mRNA (orf) at a high level in the presence of androgens. In lymphoid cells the orf-gene encodes a superantigen which has an important role in establishing self-tolerance but in mammary and breast cancer cells the function of the orf gene is unclear. In the present work we studied the expression of the S115 mammary tumor cell orf sequence and its role in the androgen regulated growth of S115 cells. The cloning and sequencing of the cDNA specific for the 1.7 kb mRNA from the S115 mouse mammary tumor cells revealed a 990 bp DNA sequence with a 99.8% homology to the Mtv-17 proviral strain. There was a difference of only one amino acid (isoleu-tyr) in the coding region. A peptide was synthesized according to the hypervariable C-terminal part of the predicted protein and used to raise a rabbit antiserum. The anti-S115-orf antiserum immunoprecipitated an approximately 45 kDa protein from the metabolically labeled S115 cell lysates. In order to analyze the putative functions of the protein, the orf-sequence was linked to MoMLV-LTR and to the human ss-actin promoter in the mammalian expression vectors pLTRpoly and pHssAPr-1-neo, respectively, and transfected into NIH3T3 and S115 cells. NIH3T3 transfectants expressing orf mRNA did not show a transformed phenotype in vitro. The S115 orf transfectants proliferated somewhat more slowly than the vector transfected control cells in cell culture, both in the presence or absence of androgen, but there was no obvious change in the phenotype of S115 cells or in expression of the fibroblast growth factor 8 (FGF-8). This factor is activated by Mtv-6 integration and mediates androgen effects in these cells. Unexpectedly, however, the formation of tumors by S115 orf cells in nude mice was considerably prolonged and tumor growth retarded when compared with vector transfected control or parent S115 cells. The results suggest that MMTV-orf can be functional in breast cancer cells but the mechanism of the growth repressive effect in mammary tumor remains to be analyzed.

Abstract: Fibroblast growth factor 8 (FGF-8) is a secreted heparin-binding protein, which has transforming potential. Alternative splicing of the mouse Fgf-8 gene potentially codes for eight protein isoforms (a-h) which differ in their transforming capacity in transfected cells. S115 mouse mammary tumor cells express a transformed phenotype and secrete FGF-8 in an androgen-dependent manner. In order to study the role of FGF-8 isoforms in the induction of transformed phenotype of breast cancer cells, we over-expressed FGF-8 isoforms a, b and e in S115 cells. Over-expression of FGF-8b, but not FGF-8a or FGF-8e, induced androgen and anchorage independent growth of S115 cells. FGF-8b-transfected S115 cells formed rapidly growing tumors with increased vascularization when injected s.c. into nude mice. FGF-8a also slightly increased tumor growth and probably tumor vascularization but FGF-8e was not found to have any effects. The angiogenic activity of FGF-8b and heparin-binding growth factor fraction (HBGF) of S115 cell conditioned media was tested in in vitro and in vivo models for angiogenesis using immortomouse brain capillary endothelial cells (IBEC) and chorion allantoic membrane (CAM) assays. Recombinant FGF-8b protein was able to stimulate proliferation, migration, and vessel-like tube formation of IBECs. In addition, stimulatory effect of S115-HBGF on IBE cell proliferation was evident. A positive angiogenic response to FGF-8b was also seen in CAM assay. The results demonstrate that the expression of Fgf-8b is able to promote vessel formation. Angiogenic capacity probably markedly contributes to the ability of FGF-8b to increase tumor growth of androgen-regulated S115 mouse breast cancer cells.

Abstract:

Abstract: Carbonic anhydrase III (CA III; EC 4.2.1.1) is a cytoplasmic enzyme that exhibits a relatively low carbon dioxide hydratase activity. It is expressed at a very high level in skeletal muscle, where physical exercise has been shown to increase free radical production. In this work we show the effect of overexpression of CA III on cellular response to oxidative stress. Rat CA III cDNA was transfected to NIH/3T3 cells, which have no endogenous CA III expression. The isolated clones expressed CA III mRNA and protein. The protein was localized to cytoplasm and nuclei. Compared to parental cells, transfected cells showed lower basal oxidized state as judged by measurement of intracellular reactive oxygen species (ROS) using fluorescent dye and an image analysis system. Addition of exogenous H2O2 to cells induced a rapid increase of ROS in control but not in CA III overexpressing cells. Association of this phenomenon with CA III expression was further confirmed by showing that overexpression of CA II could not prevent H2O2-stimulated increase of ROS. In proliferation assays, CA III overexpressing cells grew faster and were more resistant to cytotoxic concentrations of H2O2 than control cells. After a 16 h exposure to oxidative stress, the number of apoptotic cells was also reduced in transfectants. Our results suggest that CA III functions as an oxyradical scavenger and thus protects cells from oxidative damage. A lower level of free radicals in CA III overexpressing cells may also affect growth signaling pathways.

Abstract: The increase of bone resorption and reduction of bone mass in postmenopausal women can be prevented by treatment with estrogen. Although it is well established that estrogen treatment normalizes the increased bone turnover, the mechanism by which estrogen exerts its protective influence at the cellular and molecular level in bone remains elusive. It has been shown that osteoblasts are involved in osteoclast development and osteoclastic bone resorption. In this work we examine the effect of estrogen (E2) on osteoclast-mediated bone resorption via the medium conditioned by osteoblast cultures. The conditioned medium collected from osteoblast cultures without (CM) or with 0.1 nmol/L 17beta-estradiol (E-CM) was mixed in a 1:1 ratio with fresh osteoclast culture medium. Osteoclasts were isolated from the bone marrow of 3-day-old NMRI mice and cultured on bovine bone slices. The total number of multinucleated tartrate-resistant alkaline phosphatase (TRAP)-positive cells in cultures with CM and E-CM was similar to that of cells incubated in control medium. However, the number of osteoclasts containing more than three nuclei was significantly smaller in the cultures containing E-CM. The total area of resorption was only slightly decreased in cultures containing CM, but was markedly inhibited in cultures with E-CM. In osteoblast cultures, the production of interleukin (IL)-1 and IL-6, but not of TNF-alpha, was reduced by 0.1 nmol/L E2. Our data suggest that E2 treatment of osteoblasts decreases the production of factor(s) that induces osteoclast differentiation to multinucleated cells with a higher capacity for bone resorption.

Abstract: Vascular endothelial growth factor (VEGF) is a major inducer of tumor angiogenesis and an important prognostic factor in breast cancer. Hypoxia is an important inducer of VEGF expression but less is known of the role of hormones in VEGF regulation. We have studied the regulation of VEGF, VEGF-B, VEGF-C, and VEGF-D mRNAs in human MCF-7 and mouse S115 breast carcinoma cells stimulated by estrogens and androgens, respectively. VEGF, VEGF-B, and VEGF-C were expressed in both cell lines, whereas VEGF-D was expressed only in S115 cells. Addition of estradiol (E2) caused a biphasic increase of VEGF mRNA in MCF-7 cells and led to accumulation of the VEGF protein in the culture medium. The VEGF-B mRNA was not affected, while a decrease occurred in VEGF-C mRNA. Similarly, testosterone upregulated the expression of VEGF mRNA in the S115 cells. Experiments with actinomycin D and cycloheximide suggested that estrogen induction of VEGF mRNA is dependent on the synthesis of new mRNA and increased mRNA half-life. The antiestrogen ICI 182.780 inhibited E2 stimulation of VEGF, suggesting that the effect was mediated by the estrogen receptor. In contrast, the antiestrogens tamoxifen and toremifene which inhibit MCF-7 cell growth in vivo and in vitro did not inhibit estrogen effect but induced VEGF mRNA expression when used alone. The antiandrogen cyprosterone acetate inhibited T induction of VEGF mRNA in S115 cells, thus suggesting that activation of androgen receptor must be involved in the increase of VEGF mRNA. Our results suggest that both estrogen and androgen stimulate the expression of VEGF by increasing gene transcription and mRNA stability. In addition, the antiestrogens tamoxifen and toremifene also increased VEGF expression. Estrogen and androgen induction of VEGF expression and promotion of new vessel formation may be an important paracrine mechanism by which these hormones contribute to the early phase of tumor growth of hormonal cancer.

Abstract: Estrogens as well as some antiestrogens have been shown to prevent bone loss in postmenopausal women. These compounds seem to inhibit bone resorption, but their anabolic effects have been less explored. In this study, bone marrow cultures were used to compare the effect of 17beta-estradiol (E2), and two triphenylethylene derivatives, tamoxifen (TAM), and FC1271a, and a benzothiophene derivative raloxifene (RAL) on differentiation of osteoblasts. All enhanced osteoblastic differentiation of 21-day cultures as indicated by increased mineralization and bone nodule formation. All, except RAL, stimulated cell proliferation during the first 6 days of the culture. However, in the presence of RAL the content of total protein was increased in 13-day cultures. SDS-PAGE and autoradiography of [14C]-proline labeled proteins revealed elevated level of the newly synthesized collagen type I. The pure antiestrogen ICI 182,780 abolished the increase of the specific activity of alkaline phosphatase by E2, TAM, and FC1271a but not the effect of RAL on protein synthesis. Our results show that E2 as well as TAM, FC1271a, and RAL stimulate bone formation in vitro but the mechanism of the anabolic action of RAL in bone clearly differs from that of E2, TAM, and FC1271a.

Abstract: PRL is one of several polypeptide factors that regulate growth and differentiation of prostate epithelium besides steroid hormones. This hormone may also participate in the development of pathologic changes of the prostate, as evidenced by marked prostate hyperplasia in hyperprolactinemic mice. We have previously demonstrated expression of PRL receptors and androgen-dependent local production of PRL in rat and human prostate epithelium, suggesting the existence of an autocrine loop. We now show that PRL acts as a survival factor for epithelial cells of rat dorsal and lateral prostate but not ventral prostate, using long-term organ cultures as an in vitro model. Culture of prostate explants in androgen-free medium was associated with a transient surge of apoptosis during the first 2-4 days of culture in rat ventral, dorsal, and lateral prostate tissues, as quantified by either nuclear morphology or in situ DNA fragmentation analysis. PRL significantly inhibited apoptosis in androgen-deprived dorsal and lateral prostate cultures, by 40-60%, as determined by the two methods. The present study has established conditions and methodology for analysis of apoptosis in organ cultures of rat prostate and suggests a physiological role for PRL as a survival factor for prostate epithelium.

Abstract: Fgf8 is an embryonally expressed mitogenic fibroblast growth factor which has transforming capacity. It is expressed in S115 mouse mammary tumor cells (S115 cells) and in parental tumors of DD/Sio mice as well as in some human breast and prostate cancer cell lines. In S115 cells androgens induce the expression of Fgf8 which seems to be associated with the androgen-maintained malignant phenotype of the cells. S115 cells also contain and express Mtv proviruses known to insertionally activate oncogenes in other tumor cells. Here we studied the possibility of insertional activation of Fgf8 in S115 cells by MMTV proviral integration. We demonstrate by Southern blotting that the genomic DNA from DD/Sio tumors and S115 cells contains Mtv-sequences (Mtv-6 and Mtv-17) which are not found in the DNA from spleen or liver of the DD/Sio mice. In addition, the newly integrated Mtv-6 was localized to the DNA fragment containing the Fgf8 gene. Furthermore, the expression of Fgf8 mRNA in DD/Sio tumors and S115 cells was not found in mammary gland or spleen and liver of DD/Sio mice. In S115 cells, Fgf8 mRNA expression was induced in parallel to MMTV mRNA by androgen and glucocorticoids which supports the possibility that Fgf8 is controlled by the steroid-regulated MMTV-LTR. In conclusion, our data provide evidence that the insertion of MMTV into the DD/Sio tumor DNA is associated with the transcriptional activation of Fgf8 in DD/Sio tumor and consequently in S115 mouse mammary tumor cells.

Abstract: Prolactin is widely expressed in different tissues, and it is presumed to have both local and systemic actions. In males it is known to influence reproductive functions but the significance and mechanisms of prolactin action in male accessory reproductive tissues are poorly understood. Here we show that prolactin acts as a direct growth and differentiation factor for human prostate, as measured by changes in DNA synthesis and epithelial morphology of organ cultures. Furthermore, we report the expression in human prostate of a short prolactin receptor form in addition to the long form, based upon ligand cross-linking studies and RT-PCR analysis of mRNA expression. The highest density of prolactin receptors was detected in the secretory epithelial cells by immunohistochemistry. Finally, we report that prolactin is locally produced in human prostate epithelium, as evidenced by marked prolactin immunoreactivity in a significant portion of prostate epithelial cells, with parallel expression of prolactin mRNA in human prostate. Collectively, these data provide significant support for the existence of an autocrine/paracrine loop of prolactin in the human prostate and may shed new light on the involvement of prolactin in the etiology and progression of neoplastic growth of the prostate.

Abstract: Peptide hormones and growth factors are involved in the regulation of prostatic cell proliferation, differentiation, and programmed cell death, which functions are primarily controlled by androgen. In carcinogenesis, prostatic cancer cells often lose androgen dependence and become largely dependent on local growth factors. The prostatic cancer cells able to respond to factors other than androgen by proliferation and inhibition of apoptosis are possibly able to survive. We demonstrate that prostatic epithelium expresses prolactin mRNA and protein in a characteristic manner. By using in situ hybridization, an overall distribution of prolactin mRNA was demonstrated in the epithelium of rat dorsal and lateral prostate, whereas a very specific localization of prolactin protein to single cells was observed by immunohistochemistry in the same tissues. In these cells, immunoelectron microscopy showed that prolactin was primarily localized to the secretory granules. These data demonstrate a selective regulation of prostatic prolactin at least at the level of transcript processing/translation and/or protein accumulation and secretion. In addition, the expression of prolactin protein in rat dorsal and lateral prostate was found to be androgen dependent in vivo in castrated and in castrated, testosterone-treated rats, as well as in vitro in organ cultures. Our results support the concept of an autocrine/paracrine loop of prolactin action in prostate where it could mediate some of androgen actions. Also, locally synthesized prolactin might belong to the factors that take over androgen regulation of prostatic cancer cells during the development of androgen-independent growth.

Abstract: Estrogen plays an important role in the growth and maturation of bone as well as in the regulation of bone turnover in adult bone. During bone growth estrogen is needed for proper closure of epiphyseal growth plates both in females and in males. Also in young skeleton estrogen deficiency leads to increased osteoclast formation and enhanced bone resorption. In menopause estrogen deficiency induces cancellous as well as cortical bone loss. Highly increased bone resorption in cancellous bone leads to general bone loss and destruction of local architecture because of penetrative resorption and microfractures. In cortical bone the first response of estrogen withdrawal is enhanced endocortical resorption. Later, also intracortical porosity increases. These lead to decreased bone mass, disturbed architecture and reduced bone strength. At cellular level in bone estrogen inhibits differentiation of osteoclasts thus decreasing their number and reducing the amount of active remodeling units. This effect is probably mediated through some cytokines, IL-1 and IL-6 being strongest candidates. Estrogen regulates the expression of IL-6 in bone marrow cells by a so far unknown mechanism. It is still uncertain if the effects of estrogen on osteoblasts is direct or is due to coupling phenomenon between bone formation to resorption.

Abstract: Oestrogen has previously been shown to downregulate the expression of ERBB2 oncogene in human breast cancer cells, which contain a normal non-amplified ERBB2 gene. However, amplified ERBB2 seems to escape from hormonal regulation. We studied shedding of the extracellular domain (ectodomain, ECD) of the ERBB2 encoded protein in BT-474 human breast cancer cells treated with oestrogen or anti-oestrogen. Oestrogen-responsiveness of these cells has been previously demonstrated by stimulation of cell growth and expression of pS2, a marker gene known to be regulated by oestrogen receptor at transcriptional level. The concentration of the soluble ECD in the culture medium was increased by the anti-oestrogen toremifene as a function of time. In contrast, the level of ERBB2 mRNA and protein in cell lysates was not stimulated, but was transiently suppressed by toremifene. In the presence of oestrogen, the level of ECD remained low. The increased shedding of ECD in the presence of toremifene, without parallel change in ERBB2 transcripts (4.8 and 2.3 kb) and in cellular ERBB2 protein level, suggests that toremifene specifically contributes to the shedding of the ERBB2 ectodomain. These results show that shedding of ECD is an additional level of regulation of ERBB2 by the anti-oestrogen toremifene. This may contribute to resistance to growth inhibition by anti-oestrogens of breast cancers which overexpress ERBB2.

Abstract: We have studied the receptors that presumably mediate the biological effects of PRL in rat dorsal (DP) and lateral (LP) prostate. The PRL receptor proteins were localized to the glandular secretory epithelium of prostatic tissue by immunohistochemistry. Both the short and the long PRL receptor proteins were detected in DP and LP by Western blot analysis and cross-linking of [125I]human PRL to membrane preparations of DP and LP. Three messenger RNAs (mRNAs) for the long [1.3-1.7, 2.5, and 9.5-10 kilobases (kb)] and short (0.6-0.7, 3.0-4.6, and 10-12 kb) PRL receptors were expressed in dorsal and lateral lobes of rat prostate. Testosterone (T), estrogen (E), and PRL regulation of PRL receptor expression in rat DP and LP was studied in organ culture, which has been shown to be a suitable model to study hormone responses of prostatic tissue in vitro. The mRNAs of the short and long PRL receptors were differentially regulated in rat dorsolateral prostate. T, E, and PRL regulated the level of the long PRL receptor mRNAs in a tissue-specific manner, whereas hormone regulation of the short PRL receptor mRNAs was only modest. Furthermore, the hormonal responses of the different mRNA splicing variants of the long PRL receptor were not all similar; T, E, and PRL each increased the expression of 1.3- to 1.7-kb and 9.5- to 10-kb transcripts in DP, but only T did so in LP, whereas no clear regulation for the 2.5-kb mRNA could be observed in either tissue. This suggests that the hormonal regulation occurs at least at the posttranscriptional level. The effects of T and E were counteracted by the antihormones cyproterone and toremifene, respectively, indicating a specific receptor-mediated manner of steroid action.

Abstract: Inactivation of resorbing osteoclasts by calcitonin is associated with typical morphological changes and alteration of the specific organization of osteoclast cytoskeleton. Here we show that calcitonin also promotes the survival of rat osteoclasts in vitro, cultured either on glass or bone, by delaying the onset of apoptosis. Parathyroid hormone had no effect on osteoclasts cultured on glass but it slightly increased apoptosis index of osteoclasts cultured on bone. Calcitonin was also able to rescue osteoclasts in calvarial explant cultures. The survival effect of calcitonin was mimicked by dibutyryl cAMP and could not be blocked by various metabolic inhibitors known to affect the apoptotic pathway. However, clodronate-induced apoptosis of osteoclasts could not be reversed by calcitonin and neither could calcitonin rescue osteoclasts already committed to apoptosis. It did not alter the distribution of Bcl-2 in osteoclasts. Our results show that at least in vitro calcitonin protects osteoclasts from apoptosis and suggest that it regulates the onset of apoptosis.

Abstract: We studied the androgen regulation of fibroblast growth factor (FGF) receptors (FGFRs) in the Shionogi 115 (S115) mouse mammary tumor cell line and its genetic variant Clone 22. In S115 cells, androgen maintains a transformed morphology, rate of proliferation, and serum and anchorage independence. Similar effects were induced by treatment of the cells with FGF-2 or a heparin-binding growth factor (HBGF) fraction prepared from the medium conditioned by the cells. The effects of androgen and FGF-2 could be partly reversed with a specific anti-FGF-2 immunoglobulin G or by suramin, which inhibits binding of FGFs to their high affinity receptors. Testosterone and FGF-2 increased the expression of FGFR-1 messenger RNA (mRNA) and, to a lesser extent, FGFR-3 mRNA, but down-regulated FGFR-2 mRNA in S115 cells. No FGFR-4 mRNA was detected. FGF-2 also down-regulated the expression of syndecan-1, a heparan sulfate proteoglycan that binds FGF with low affinity. The binding of radiolabeled FGF-2 to FGFRs was lower in the cells cultured with testosterone or in the presence of the HBGFs from androgen-treated cells, presumably because of the autocrine production of FGF-like factors. In Clone 22 cells, FGFRs and syndecan-1 responded to androgen as in S115 cells, but they were less sensitive to FGF-2. Androgen or FGF-2 could not induce morphological transformation, although both stimulated proliferation. Androgen-increased proliferation was not, however, decreased by anti-FGF-2 immunoglobulin G in Clone 22 cells. These data suggest that of the HBGFs produced, FGF-2 is required in androgen induction of morphological change, whereas the effect on proliferation involves other factors as well (perhaps mostly FGF-8). The results show that androgen differentially regulates the expression of the high and low affinity FGF receptors, which could mediate androgen induction of the transformed phenotype in S115 cells by an autocrine mechanism. The differential responses of the Clone 22 variant cells to androgen and FGF-2 suggest that the pathways of steroid induction of different parameters of the transformed phenotype, such as transition to fibroblastic morphology and stimulation of proliferation, are divergent.

Abstract: BACKGROUND: Antiestrogens inhibit the stimulative effects of estrogens on breast cancer growth, but the mechanism(s) by which they trigger tumor regression are not completely understood. Growth retardation and tumor regression can be achieved by enhanced cell death and/or arrested cell proliferation. PURPOSE: Our aim was to investigate the effect of a new antiestrogen, toremifene, on human breast cancer cells grown either in culture or as tumors in nude mice. METHODS: The growth and morphology of in vitro cultured cells of the human breast cancer cell line MCF-7 were monitored by time-lapse video. MCF-7 cells and ZR-75-1 human breast cancer cells were grown as tumors in nude mice and subsequently examined by electron microscopy. The integrity of DNA isolated from these cells was determined by standard gel electrophoretic techniques. Northern blot hybridization analysis was used to determine the steady-state levels of the mRNAs for testosterone-repressed prostatic message-2 (TRPM-2), tumor growth factor beta-1 (TGF beta 1), and pS2 (a small, cysteine-rich protein of unknown function). RESULTS: Time-lapse video microscopy of the cell cultures indicated that treatment with 7.5 microM toremifene for 3 days caused approximately 60% of the cells to exhibit morphologic characteristics typical of cells undergoing programmed death, or apoptosis. The number of mitoses gradually decreased to zero over a 3- to 4-day period. Estrogen withdrawal for the same length of time resulted in an approximately equal number of apoptoses and mitoses. These changes were not associated with the pattern of DNA fragmentation, detectable as ladders in agarose gels, that is characteristic of the DNA of cells undergoing apoptosis. Elevated levels of TRPM-2 and TGF beta 1 mRNAs were observed in in vitro or in vivo grown tumor cells treated with 5-10 microM toremifene. Elevated levels of TRPM-2, but not TGF beta 1, mRNA were observed in the tumor cells after estrogen withdrawal. The steady-state level of pS2 mRNA in the tumor cells dropped in response to either toremifene treatment or estrogen withdrawal. CONCLUSION: Toremifene causes growth inhibition of estrogen-sensitive breast cancer cells by inducing some cells to undergo apoptosis and by inhibiting other cells from entering mitosis. The higher than normal amounts of TRPM-2 and TGF beta 1 protein that would likely result from the elevated levels of TRPM-2 and TGF beta 1 mRNAs measured in these cells after toremifene treatment may have an important role in the growth inhibition process. IMPLICATION: Apoptosis as an active, targeted process provides a potential new therapeutic approach for treating breast cancer.

Abstract: We have established organ cultures of human prostate for in vitro analysis of the hormone responsiveness of prostatic carcinoma. Tissue samples were obtained from total prostatectomies for localized cancer. Normal prostate tissues with age-related hyperplastic changes were obtained from cystoprostatectomies of bladder cancer patients representing the same age group, and they wer cultivated as controls. The explants of prostates were cultured for 7 days in basal medium containing 5% dextran charcoal-treated fetal calf serum, insulin (0.08 IU/ml), and dexamethasone (10(-7) M) with or without dihydrotestosterone (DHT) (10(-7) M) or estradiol (10(-9) M). Control prostates showed involutive changes of morphology when cultured in basal medium. These changes were prevented by DHT, which also maintained a strong epithelial immunostaining for PSA (prostate specific antigen), which was used as a marker for tissue-specific functions. The concentration of PSA in the medium was high. The rate of [3H]thymidine incorporation into DNA was stimulated by DHT in some cultures of control prostates, but no increase was seen in the others. Androgen stimulation of [3H]thymidine incorporation was consistently inhibited by the antihormone cyproterone acetate. The main morphological response of cultured control prostates to estradiol was induction of squamous metaplasia. This was associated with increased incorporation of [3H]thymidine, which was radioautographically localized to the basal layer of epithelium. Estradiol effects were counteracted by the antihormone toremifene. The expression of androgen receptor mRNA and protein in cultured control prostate was demonstrated by Northern blotting and immunohistochemistry, respectively. Also, the expression of estrogen receptor was demonstrated by the polymerase chain reaction analysis of total mRNA from cultured control and cancer prostate. The cultured explants of prostate cancer maintained the overall morphology of the original carcinoma. However, the presence of DHT improved the morphology of cancerous acini in all better differentiated carcinomas (3 grade I and 5 grade II), and corresponding responses to DHT were observed in the rate of DNA labeling with [3H]thymidine. In 2 of 3 grade I carcinomas, DHT increased DNA synthesis, but in grade II cancers the patterns of hormone responses were more variable. The poorly differentiated grade III prostatic carcinomas did not respond to either hormone as measured by [3H]thymidine uptake, and no hormone effects could be seen in morphology. Immunostaining for PSA differed from that in control prostates: besides cancerous acini, the surrounding stroma was also intensively stained, which suggests unpolarized and impaired secretion of PSA by the cancer cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Abstract: Hormone regulation of carbonic anhydrase II (CA II) was studied in rat dorsal and lateral prostate. CA II is a major soluble protein in these accessory sex glands. The immunoelectronmicroscopy showed that CA II is expressed in their epithelial cells only. For studies on hormone regulation, adult male rats were castrated for 2 or 7 days. Groups of 7-day castrates and normal rats were treated daily either with testosterone or 17-beta-estradiol for 6 days and 2-day castrates for 1 day. CA II protein was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and quantified by RIA. The levels of CA II mRNA were studied by Northern blotting and hybridization of total RNA with a 32P-labeled mouse CA II cDNA clone. Castration of the rats decreased the concentration of CA II in lateral prostate but increased in dorsal prostate. These changes were reversed in both prostatic lobes by testosterone treatment. Estrogen treatment of castrated rats enhanced CA II concentration in lateral prostate but no effects were seen in the dorsal prostate of the same animals. In normal rats estrogen increased CA II concentration of dorsal prostate but there was no change in lateral prostate. Corresponding changes were observed in the levels of CA II mRNA in both tissues. The morphometric analyses showed that the castration- and hormone-induced changes of the mRNA and protein levels of the exclusively epithelial CA II could not be explained by any alterations in the proportions of epithelial and stromal components of the glands after hormone manipulations. The results demonstrate the differential steroid regulation of CAII in two prostatic lobes. Androgen regulates the expression of CAII at messenger RNA level, but the responses of CAII to testosterone are opposite in dorsal and lateral prostate. Estrogen increases CA II expression in lateral prostate but in dorsal prostate the castration-like effects of estrogen on CAII expression are probably indirect.

Abstract: Besides androgens, estrogen (E) and PRL are thought to have important roles in the regulation of the growth and function of the prostate. We have established organ cultures of rat dorsolateral prostate for the analysis of the multiple hormone actions. Explants of dorsal prostate (DP) and lateral prostate (LP) were cultured in a serum-free basal medium containing insulin and corticosterone with or without the hormones studied. The viability and overall integrity of the tissues were maintained for at least 14 days. The morphology of the explants showed castration-like changes in the basal medium, but the addition of testosterone (T) prevented them. Androgen receptors in the prostate cultured with T were demonstrated by immunohistochemistry. When the explants were grown with E the epithelium became stratified, and the cells were flat. The epithelium was also layered when the explants were grown with PRL, but the epithelial cells were hypersecretory and large. The glandular morphology of the cultured prostate was, however, best preserved if T was added along with E or PRL. The wet weights and DNA contents of the explants declined during the culture, but they were better maintained if T, E, or PRL were added to the medium. The rate of DNA labeling with [3H]thymidine was activated in the cultured explants, but it was higher in those grown with T, E, or PRL than in those grown in the basal medium. The tissue specific functions were evaluated by measuring the expression of the genes RWB and M-40.3 encoding androgen-regulated secretory proteins. The steady state levels of RWB and M-40.3 mRNA were low in the explants grown in the basal medium but in the presence of T they were high. E and PRL also increased the expression of RWB and M-40.3 messenger RNA, although the responses in DP and LP were somewhat different. The antihormones cyproterone and toremifene opposed the increase of M-40.3 messenger RNA by T and E, respectively. The results show that the cultured DP and LP of the rat maintain the androgen responsiveness and tissue-specific functions in vitro. In addition, E and PRL have androgen-independent, direct effects in them. Rat dorsolateral prostate in culture thus provides a useful model for the studies on the mechanisms of hormone regulation of the prostate.

Abstract: Amplification and enhanced expression of the erbB2/HER-2/neu gene has been associated with an increased growth rate and poor prognosis of human breast cancer. We have studied the relationship between erbB2 expression and the regulation of cell growth by estrogen and anti-estrogens in the human breast cancer cell line ZR-75-1 in vitro and in athymic nude mice, pS2 being used as a marker gene for estrogen-stimulated gene expression. Only low amounts of erbB2 mRNA were seen in the cells grown in vitro in the presence of estrogen which stimulated the cells to proliferate rapidly and induced the expression of pS2 mRNA. Upon hormone withdrawal, erbB2 mRNA and protein increased, while pS2 mRNA declined to an undetectable level and cell proliferation slowed down. Opposite but more rapid changes were observed upon estrogen addition. The anti-estrogens toremifene and tamoxifen inhibited estrogen induction of pS2 expression, down-regulation of erbB2 expression and proliferation of the ZR-75-I cells in a concentration-dependent manner. Similar results were obtained in nude mice. ZR-75-I cells formed tumors only in mice carrying estrogen pellets. In these tumors little erbB2 mRNA was seen. Concomitant administration of toremifene or tamoxifen increased erbB2 mRNA and abolished pS2 mRNA. Our results show that enhanced expression of erbB2 is associated with hormone deprivation and growth arrest of the estrogen-dependent breast cancer cell line ZR-75-I. Thus, in mammary epithelial cells, erbB2 may have important estrogen-regulated functions which are not related to cell proliferation.

Abstract: We have generated temperature-sensitive (ts) mutants for steroid-regulated anchorage-independent cell growth. Androgen-responsive S115+A mouse mammary tumor cells were mutagenized with ethyl methane sulfonate and the variants which were growth-arrested in suspension at the nonpermissive temperature of 41 degrees C were selected by killing dividing wild-type cells with the DNA synthesis inhibitors 5-fluoro-2'-deoxyuridine or cytosine arabinoside. Fifteen clones were isolated and characterized for morphology and growth properties. Three (ts21, ts27, ts33) of the phenotypic variants were ts for androgen-maintained anchorage-independent growth, two of them (ts27 and ts33) also for growth in monolayer. Growth arrest at 41 degrees C was not due to a defect in androgen receptor function in any of the mutant cell lines as shown by steroid binding assays and by the androgen-stimulated expression of both endogenous MMTV RNA and the transiently transfected LTR-CAT gene at the nonpermissive temperature. It remains to be determined for clone ts33 whether the defect is in postreceptor events of steroid action or in genes affecting general mechanisms of cell growth. However, since in clones ts21 and ts27 general cell growth remains functional at 41 degrees C under serum stimulation, defects may be in postreceptor steroid-related pathways. It is hoped that these mutants will provide a useful tool for study of steroid regulation of cell growth and in particular of the property of anchorage-independent growth.

Abstract: The distribution of carbonic anhydrase isoenzymes I and II in rat accessory sex glands was studied by use of immunohistochemistry. Carbonic anhydrase I could not be demonstrated in any of the organs studied, whereas carbonic anhydrase II was found exclusively in epithelial cells of the lateral and dorsal prostates as well as in epithelial cells of seminal vesicles and the coagulating gland. In contrast, the ventral prostate contained neither isoenzyme. Sodium dodecyl sulfate-slab electrophoresis and immunoblotting experiments suggested that carbonic anhydrase II is a major soluble protein in the lateral prostate. This was confirmed by radioimmunoassay, which showed that carbonic anhydrase II represents about 15 percent of soluble proteins in the rat lateral prostate. It was further demonstrated that the relative amount of carbonic anhydrase II in the rat lateral prostate and seminal vesicles is under testosterone regulation. It is suggested that carbonic anhydrase II in rat accessory sex glands is involved in bicarbonate production, a function that particularly characterizes the rat lateral prostate.

Abstract: The organ culture of the rat ventral prostate was chosen as a model to determine whether any of the estrogen effects in vivo on the prostate are direct and expressed at the hormone concentrations normally found in the male. During 2 weeks of culture, estradiol at the high concentration of 10(-5) M blocked the androgenic activation of [3H]thymidine incorporation into DNA. The inhibition was localized in epithelium. Protein content of testosterone-treated explants and the accumulation of prostatein in the medium were considerably decreased, indicating inhibition of secretion. Antiandrogenic effects were not seen in morphology of estrogen-treated explants. The lower concentrations (from 10(-9) M to 10(-6) M) of estradiol increased the volume density of epithelium from day 7 onwards. The height of epithelium was concomitantly increased. The volume density of epithelium as well as the percentage of acini with metaplastic changes were significantly increased. These epithelial changes were less pronounced in the presence of androgen, suggesting that physiological concentrations of androgen prevent the expression of estrogen action in the morphology of the prostate. A change in staining with peanut (PNA)- and wheat germ agglutinin (WGA)-lectins indicated defective secretory capacity in metaplastic epithelium. In spite of the increased protein content in the explants, no constant pattern of the changes in prostatein accumulation could be recorded. Although the concentrations of estrogen required to induce squamous metaplasia were still unphysiological, the occurrence of this abnormal differentiation of the prostatic epithelium suggests that the cooperative action of estrogen is involved in androgen-dependent normal epithelial growth and possibly also in promoting growth of prostatic neoplasia.

Abstract: The effect of glucose on the androgen-maintained protein synthesis was studied in the cultured rat ventral prostate. The explants were cultivated for 5 days in the glucose-free medium containing 10% fetal calf serum with or without 10 mM glucose and 10(-7) M testosterone. In some experiments tunicamycin, a specific inhibitor of protein glycosylation was added to the glucose-containing medium. The morphological integrity of the tissue was maintained in all the mediums used. At the end of the culture, the explants were incubated with [35S]methionine. Soluble radioactive proteins were separated by the SDS-polyacrylamide gel electrophoresis and analyzed further by the fluorography. Glucose was necessary for the testosterone-maintained accumulation of three components (Mr less than 14,000) of the major prostatic secretory protein. The electrophoretic migration, glycosylation pattern and immunological data (not shown) indicated that it was the well-known prostatic binding protein. On the other hand, two prominent polypeptides (Mr 70,000 and 100,000) appeared in the absence of glucose. Glucose starvation and the inhibition of glycosylation with tunicamycin caused similar effects on the labelling of the newly-synthesized soluble proteins. The mechanisms of glucose maintenance of the major prostatic protein and suppression of two high molecular weight proteins seemed to be different, although glycosylation was probably involved in both glucose effects.

Abstract: Androgenic control of citrate metabolism was studied by measuring the conversion of (2-14C)acetate or (6-14C)glucose to (14C)citrate and 14CO2 in the ventral prostate of the rat. The decarboxylation of (2-14C)acetate showed that androgen preferentially increased (14C)citrate oxidation, probably to meet the increased energy demands of cellular synthetic reactions. This led to the decreased accumulation of (14C)citrate from (2-14C)acetate. On the other hand, both the production of (14C)citrate and the formation of 14CO2 from (6-14C)glucose were decreased by castration and increased by testosterone, this being mainly due to the androgenic control of pyruvate dehydrogenase. These changes were more marked and rapid than those in oxygen consumption, in (2-14C)acetate oxidation, or in the total content of prostatic citrate that was maintained by testosterone. Glucose as the main source of citrate in testosterone-treated rats can thus be replaced by alternative substrates in castrated rats. The rate of citrate accumulation could be more dependent on the number of secretory cells than their hormonal activation.