Estrogen responses in bovine fetal uterine cells involve pathways directed by both estrogen response element and activator protein-1.
(57/8473)
Objectives were to examine possible roles of estrogen receptor (ER) in development of the bovine uterine endometrium in the context of ER type, enhancer type, and ligand-independent activation. Expression vectors producing either ERalpha or ERbeta were introduced into fetal uterine cells from Day 110 to 120 of gestation (UBF120 cells) and into rat embryo fibroblasts (Rat-1 cells), neither of which express endogenous ER. Reporter constructs containing either an estrogen response element (ERE) or activator protein-1 (AP-1) response element were cotransfected. These reporters were also transfected into fetal uterine cells from Day 180 to 200 of gestation (UBF180 cells), which express ER. In UBF120 and Rat-1 cells transfected with either ERalpha or ERbeta, treatment with estradiol-17beta (E2) resulted in increased activity of an ERE reporter construct, but not an AP-1 element reporter construct. The antiestrogen ICI 182,780 (ICI) exhibited E2 antagonist activity with both ERalpha and ERbeta. Thus, all components were present for E2-dependent transcription from an ERE except ER; however, cells were not competent for E2-dependent transcription mediated through AP-1. In UBF180 cells, E2 treatment increased both ERE and AP-1 reporter activity. ICI exhibited E2 antagonist activity. Treatment with epidermal growth factor resulted in increased ERE reporter activity that was inhibited by ICI, indicative of ligand-independent activation of ER. These data suggest that multiple pathways for ER-mediated gene regulation occur in the developing fetal uterus and that nuclear components necessary for action of both ERalpha and ERbeta are present prior to expression of the receptor. (+info)
Severe combined immunodeficient-hu model of human prostate cancer metastasis to human bone.
(58/8473)
Commonly used in vivo models of prostate cancer metastasis include syngeneic rodent cancers and xenografts of human cancer in immunodeficient mice. However, the occurrence of osseous metastases in these models is rare, and in xenograft models, species-specific factors may limit the ability of human cells to metastasize to rodent bones. We have modified the severe combined immunodeficient (SCID)-human model to test the ability of circulating human prostate cancer cells to home to macroscopic fragments of human bone and other organs previously implanted into SCID mice. We have also compared the growth of human prostate cancer cells in various human and mouse tissue microenvironments in vivo. Macroscopic fragments of human fetal bone, lung, or intestine (16-22 weeks gestation) or mouse bone were implanted s.c. into male CB.17 SCID mice. Four weeks later, human prostate cancer cells were injected either i.v. via the tail vein (circulating cell colonization assay) or directly into the implanted tissue fragments transdermally (end organ growth assay). Tumor growth was followed for 6 weeks by palpation and magnetic resonance imaging. After 6 weeks, tumors were enumerated in implanted human and mouse organ fragments and native mouse tissue. Tumors were characterized by histology, immunohistochemistry, and chromosomal analysis. After i.v. injection, circulating PC3 cells successfully colonized implanted human bone fragments in 5 of 19 mice. Tumors were easily followed by palpation and imaging and had an average volume of 258 mm3 at autopsy. Histological examination revealed osteolysis and a strong desmoplastic stromal response, which indicated intense stromal-epithelial interaction. Bone tumors were subcultured, and chromosomal analysis demonstrated that the tumors were derived from the parental prostate cancer cell line. Microscopic tumor colonies were also found in a few mouse lungs after i.v. injection of PC3, DU145, and LNCaP cells, however the volume of the lung nodules was less than 1 mm3 in all of the cases. No colonization of human lung or intestine implants, the mouse skeleton, or other mouse organs was detected, demonstrating a species- and tissue-specific colonization of human bone by PC3 cells. Direct injection of 10(4) prostate cancer cells into human bone implants resulted in large tumors in 75-100% of mice. PC3 and DU145 bone tumors were primarily osteolytic, whereas LNCaP bone tumors were both osteoblastic and osteolytic. PC3 and LNCaP bone tumors showed a desmoplastic stromal response, which indicated intense stromal-epithelial interaction. All three of the cell lines formed tumors in implanted human lung tissue; however, the tumors were all < or = 10 mm3 in volume and showed minimal stromal involvement. No tumors formed after either s.c. injection or injection of cells into implanted mouse bone demonstrating both species- and tissue-specific enhancement of growth of human prostate cancer cells by human bone. The severe combined immunodeficient-human model provides a useful system to study species-specific mechanisms involved in the homing of human prostate cancer cells to human bone and the growth of human prostate cancer cells in human bone. (+info)
Commitment to the monocytic lineage occurs in the absence of the transcription factor PU.1.
(59/8473)
Mice homozygous for the disruption of the PU.1 (Spi-1) gene do not produce mature macrophages. In determining the role of PU.1 in macrophage differentiation, the present study investigated whether or not there was commitment to the monocytic lineage in the absence of PU.1. Early PU.1-/- myeloid colonies were generated from neonate liver under conditions that promote primarily macrophage and granulocyte/macrophage colonies. These PU.1-/- colonies were found to contain cells with monocytic characteristics as determined by nonspecific esterase stain and the use of monoclonal antibodies that recognize early monocyte precursors, including Moma-2, ER-MP12, ER-MP20, and ER-MP58. In addition, early myeloid cells could be grown from PU.1-/- fetal liver cultures in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF). Similar to the PU.1 null colonies, the GM-CSF-dependent cells also possessed early monocytic characteristics, including the ability to phagocytize latex beads. The ability of PU.1-/- progenitors to commit to the monocytic lineage was also verified in vivo by flow cytometry and cytochemical analysis of primary neonate liver cells. The combined data shows that PU.1 is absolutely required for macrophage development after commitment to this lineage. (+info)
Mitigation of caffeine-induced fetopathy in mice by pretreatment with beta-adrenergic blocking agents.
(60/8473)
In a previous experiment, fetopathic effects of caffeine were significantly reduced by pretreatment with propranolol at dosage levels of 2.5 to 10 mg/kg. The present experiments were undertaeken to investigate the relation between time intervals of propranolol pretreatment and its effect on reducing fetopathy. Furthermore, the effect of timolol, another beta-adrenergic blocking agent, on reducing fetopathy was compared with that of propranolol. Propranolol (5 mg/kg) administered 15, 30 or 60 minutes before caffeine treatment significantly reduced the caffeine-induced fetopathy. The optimal effect was found when propranolol was given 30 minutes before caffine. The reduction in fetopathy by timolol pretreatment was comparable to that of propranolol. The results lend support to the hypothesis that the fetopathic effect of caffeine is linked with released catecholamines in material or fetal issues of mice. (+info)
Amino acid composition of the fetal pig.
(61/8473)
Amino acid composition and accretion were determined in fetal pigs obtained from gilts by hysterectomy at d 40-114 of gestation. The whole homogenate of the fetal pig was used for analysis of dry matter, nitrogen and amino acids. Uterine uptake of amino acids was estimated at d 110-114 of gestation on the basis of uterine arteriovenous concentrations. Nitrogen and amino acid accretion in fetal pigs increased more rapidly with gestation than non-nitrogen dry matter. Amino acid nitrogen represented 83-88% of total nitrogen, and arginine was the most abundant nitrogen carrier in fetal pigs at all gestational ages. Amino acid composition changed with gestation, with glycine and hydroxyproline increasing (P < 0.05) markedly and other amino acids (except ornithine and tryptophan) decreasing (P < 0.05) to a lesser extent. Amino acid concentrations in fetal pigs increased (P < 0.05) progressively from d 60 to 114 of gestation. Uterine uptake of arginine and proline plus hydroxyproline met requirements for fetal growth during late gestation only marginally, and uterine uptake of aspartate/asparagine and glutamate was only 9-29% of fetal accretion. In contrast, uterine uptake of citrulline and ornithine was 55- and 15-fold greater (P < 0.05) than fetal accretion, respectively. On the basis of hydroxyproline content, collagen was estimated to represent approximately 7, 15, 25, 28 and 29% of total body protein at d 40, 60, 90, 110 and 114 of gestation, respectively. Amino acid composition of the fetal pig is similar to that for the human fetus, indicating that the pig is an excellent model for studying amino acid nutrition and metabolism in the human preterm neonate and infant. (+info)
Direct and indirect modulation of ornithine decarboxylase and cyclooxygenase by UVB radiation in human skin cells.
(62/8473)
Exposure to solar ultraviolet (UV) B radiation is responsible for skin inflammation and tumour progression. Cyclooxygenase and ornithine decarboxylase are believed to be involved in such processes since they participate in the synthesis of mediators of inflammation and cell differentiation, respectively. We have investigated the in vitro modulation of expression of such genes by UVB radiation in different skin cell lines. We have observed that accumulation of ornithine decarboxylase mRNA is unaffected by even high UVB doses in both human epidermal keratinocytes and dermal fibroblasts, whereas cyclooxygenase-2 levels were significantly up-regulated by low UVB doses in KB human epidermoid keratinocytes. Depletion of total intracellular glutathione levels in KB cells amplified the activation, revealing a role for an oxidative component of UVB in modulating cyclooxygenase gene expression. Transfer of medium from UVB irradiated keratinocytes to fibroblasts resulted in a significant activation of cyclooxygenase expression and activity, while ornithine decarboxylase levels were unaffected. We conclude that UVB radiation can activate cyclooxygenase gene expression in human skin cells both by direct activation pathways or indirectly by inducing a paracrine mechanism. (+info)
Fetal rat adrenal steroidogenesis and steroid transfer to adrenalectomized mother.
(63/8473)
On the 22nd day of gestation in rats, fetuses of acutely adrenalectomized mothers were injected subcutaneously with 0.43 muCi 4-14C-progesterone in 0.05 ml saline. Ten and 20 min after injection to fetuses, samples were taken to determine the 14C-progesterone metabolites in the plasma and adrenal glands. After extraction of the samples taken, the metabolites were separated by two-dimensional thin-layer chromatography and identified by autoradiography. 11-deoxycorticosterone, 18-hydroxy-11-deoxycorticosterone, corticosterone and 11beta-hydroxyprogesterone were identified in the plasma of injected fetuses, and, in far smaller amounts, in the plasma of their mothers. The plasma of noninjected fetuses also contained very small amounts of these corticoids. The fetal adrenal glands contained far smaller amounts of radioactive steroids than the fetal plasma did. The results obtained show that steroids of fetal origin can cross the placenta in and out, constituting evidence that the fetal adrenal glands are the only source of the plasma corticoids of their adrenalectomized mothers. (+info)
Dynamic association of proteasomal machinery with the centrosome.
(64/8473)
Although the number of pathologies known to arise from the inappropriate folding of proteins continues to grow, mechanisms underlying the recognition and ultimate disposition of misfolded polypeptides remain obscure. For example, how and where such substrates are identified and processed is unknown. We report here the identification of a specific subcellular structure in which, under basal conditions, the 20S proteasome, the PA700 and PA28 (700- and 180-kD proteasome activator complexes, respectively), ubiquitin, Hsp70 and Hsp90 (70- and 90-kD heat shock protein, respectively) concentrate in HEK 293 and HeLa cells. The structure is perinuclear, surrounded by endoplasmic reticulum, adjacent to the Golgi, and colocalizes with gamma-tubulin, an established centrosomal marker. Density gradient fractions containing purified centrosomes are enriched in proteasomal components and cell stress chaperones. The centrosome-associated structure enlarges in response to inhibition of proteasome activity and the level of misfolded proteins. For example, folding mutants of CFTR form large inclusions which arise from the centrosome upon inhibition of proteasome activity. At high levels of misfolded protein, the structure not only expands but also extensively recruits the cytosolic pools of ubiquitin, Hsp70, PA700, PA28, and the 20S proteasome. Thus, the centrosome may act as a scaffold, which concentrates and recruits the systems which act as censors and modulators of the balance between folding, aggregation, and degradation. (+info)