Expression and up-regulation of retinoic acid receptor-beta is associated with retinoid sensitivity and colony formation in esophageal cancer cell lines.
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Retinoids exhibit chemotherapeutic and chemopreventive activities, possibly due to their ability to modulate cell growth, differentiation, and apoptosis. These effects are thought to be mediated by nuclear retinoic acid (RA) receptors (RARs) and retinoid X receptors, each of which includes three subtypes (alpha, beta, and gamma) that act as transcription factors. To determine whether RARs play a role in mediating the effects of RA on human esophageal cancer (HEC) cells, we analyzed the effects of RA on: (a) the growth, differentiation, and apoptosis in seven HEC cell lines; (b) receptor expression; (c) receptor modulation by RA; and (d) expression of receptors in 20 surgical HEC specimens. RA inhibited the growth of five of seven cell lines and also the constitutive expression of the squamous differentiation markers cytokeratin 1 and transglutaminase I in all cell lines. The growth inhibition by RA was due to the induction of apoptosis in the five cell lines. All seven cell lines expressed RAR-alpha and RAR-gamma, and four cell lines showed some changes by RA, but not associated with apoptosis. In contrast, RAR-beta was expressed in five of seven cell lines and up-regulated by RA in these five cell lines, which were associated with apoptosis. Two cell lines that failed to express RAR-beta showed no growth inhibition or apoptosis and no RAR-beta inducibility. Interestingly, only these two cell lines were able to form colonies in soft agar. RAR-alpha, RAR-beta, and RAR-gamma mRNAs were expressed in all 20 adjacent normal esophageal tissues. The expression of RAR-alpha and RAR-gamma remains positive in HEC specimens, but RAR-beta expression was detected in only 6 of 20 HEC specimens. These data suggest that the expression of RAR-beta is associated with response of HEC cells to RA and that the loss of RAR-beta expression may be associated with HEC development. (+info)
Regression of hypertrophied rat pulmonary arteries in organ culture is associated with suppression of proteolytic activity, inhibition of tenascin-C, and smooth muscle cell apoptosis.
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Increased elastase activity and deposition of the matrix glycoprotein tenascin-C (TN), codistributing with proliferating smooth muscle cells (SMCs), are features of pulmonary vascular disease. In pulmonary artery (PA) SMC cultures, TN is regulated by matrix metalloproteinases (MMPs) and mechanical stress. On attached collagen gels, MMPs upregulate TN, leading to SMC proliferation, whereas on floating collagen, reduced MMPs suppress TN and induce SMC apoptosis. We now investigate the response of SMCs in the whole vessel by comparing attached and floating conditions using either normal PAs derived from juvenile pigs or normal or hypertrophied rat PAs that were embedded in collagen gels for 8 days. Normal porcine PAs in attached collagen gels were characterized by increasing activity of MMP-2 and MMP-9 assessed by zymography and TN deposition detected by Western immunoblotting and densitometric analysis of immunoreactivity. PAs on floating collagen showed reduced activity of both MMPs and deposition of TN. Tenascin-rich foci were associated with proliferating cell nuclear antigen immunoreactivity, and TN-poor areas with apoptosis, by terminal deoxynucleotidyl transferase-mediated nick end labeling assay, but no difference in wall thickness was observed. Although normal rat PAs were similar to piglet vessels, hypertrophied rat PAs showed an amplified response. Increased elastase, MMP-2, TN, and elastin deposition, as well as SMC proliferating cell nuclear antigen positivity, correlated with progressive medial thickening on attached collagen, whereas reduced MMP-2, elastase, TN, and induction of SMC apoptosis accompanied regression of the thickened media on floating collagen. In showing that hypertrophied SMCs in the intact vessel can be made to apoptose and that resorption of extracellular matrix can be achieved by inhibition of elastase and MMPs, our study suggests novel strategies to reverse vascular disease. (+info)
Significance of myocytes with positive DNA in situ nick end-labeling (TUNEL) in hearts with dilated cardiomyopathy: not apoptosis but DNA repair.
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BACKGROUND: The presence of apoptotic myocytes has been reported in human hearts with dilated cardiomyopathy (DCM) on the basis of a positive finding of DNA in situ nick end-labeling (TUNEL). However, ultrastructural evidence of myocyte apoptosis has not been obtained. METHODS AND RESULTS: A total of 80 endomyocardial biopsies were obtained from right and left ventricles of 20 patients with DCM and 20 normal control subjects. TUNEL-positive myocytes were found by light microscope in 15% of DCM specimens (controls, 0%, P<0.05), and the percentage of TUNEL-positive myocytes per section in DCM was 1. 0+/-2.7% (mean+/-SD). According to TUNEL at the electron microscopic level (EM-TUNEL), immunogold particles, which label DNA breaks with 3'-OH terminals, were markedly accumulated in the bizarre-shaped nuclei, with widespread clumping of chromatin (so-called "hypertrophied nuclei") of the myocytes obtained from DCM. Their ultrastructure was neither apoptotic nor necrotic but rather that of living cells. Taq polymerase-based DNA in situ ligation assay, which detects double-stranded DNA fragments more specifically than TUNEL, did not detect a positive reaction in any case. In mirror sections, all of the TUNEL-positive myocytes in DCM simultaneously expressed proliferating cell nuclear antigen, which is required for both DNA replication and repair, but Ki-67, a replication-associated antigen, was completely negative in all cases, which appeared to rule out cell proliferation activity. CONCLUSIONS: Most of the TUNEL-positive myocytes in hearts with DCM are not apoptotic but rather living cells with increasing activity of DNA repair. (+info)
Mechanisms of apoptosis in T cells from patients with renal cell carcinoma.
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Tumors may escape immune recognition and destruction through the induction of apoptosis in activated T lymphocytes. Results from several laboratories suggest that FasL (L/CD95L) expression in tumors may be responsible for this process. In this study of patients with renal cell carcinoma (RCC), we provide evidence for two mechanisms of T-cell apoptosis. One mechanism involves the induction of apoptosis via FasL expression in tumor cells. This is supported by several observations, including the fact that tumor cells in situ as well as cultured cell lines expressed FasL mRNA and protein by a variety of techniques. The FasL in RCC is functional because in coculture experiments, FasL+ tumors induced apoptosis in Fas-sensitive Jurkat T cells and in activated peripheral blood T cells but not in resting peripheral blood T cells. Most importantly, antibody to FasL partially blocked apoptosis of the activated T cells. Moreover, Fas was expressed by T cells derived from the peripheral blood (53% median) and tumor (44.3% median) of RCC patients. Finally, in situ staining for DNA breaks demonstrated apoptosis in a subset of T cells infiltrating renal tumors. These studies also identified a second mechanism of apoptosis in RCC patient peripheral T cells. Whereas these cells did not display DNA breaks when freshly isolated or after culture for 24 h in medium, peripheral blood T cells from RCC patients underwent activation-induced cell death after stimulation with either phorbol 12-myristate 13-acetate/ionomycin or anti-CD3/CD28 antibodies. Apoptosis mediated by exposure to FasL in tumor cells or through T-cell activation may contribute to the failure of RCC patients to develop an effective T-cell-mediated antitumor response. (+info)
Fas receptor up-regulation and membrane localization concurrent with apoptosis in idiopathic esophageal ulceration: toward a better understanding of esophageal injury in AIDS.
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Activation of the Fas-mediated apoptotic pathway in the etiology of idiopathic esophageal ulcerations (IEUs) was investigated. Constitutive expression of Fas ligand (Fas L) was found in the basal layer of the normal esophageal mucosa and in IEUs and cytomegalovirus ulcerations. In addition to an altered cytokine environment, there was significant up-regulation of Fas antigen mRNA and membrane localization of the receptor in IEU specimens. Concomitant increase in the basic components of Fas machinery, together with elevated mucosal apoptosis, strongly suggests the potential role of the Fas signaling pathway in the onset of tissue destruction associated with IEUs. (+info)
Effects of neonatal diethylstilbestrol (DES) exposure on morphology and growth patterns of endometrial epithelial cells in CD-1 mice.
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The effects of neonatal diethylstilbestrol (DES) exposure on the morphology and proliferating patterns of endometrial epithelial cells were investigated at various stages of development in mice. Female CD-1 mice were given daily subcutaneous injections of 2 micrograms of DES in corn oil or corn oil alone (control) at 1-5 days of age and were killed at 5, 6, 7, 8, 15, and 22 days of age. At 5 days of age, the uteri of DES-treated mice had expanded lumina and undulated luminal surfaces lined by slightly elongated epithelial cells. At 6-8 days of age, marked infolding of clusters of hypertrophic elongated luminal epithelial cells was present; uteri had disorganized endometrial stromal and myometrial layers. At 15 and 22 days of age, the tissues from DES-treated mice had decreased numbers of endometrial glands, minimal stromal fibrosis, and smaller uterine horns than did the controls. Ultrastructurally, the endometrial epithelial cells of DES-treated mice at 5 and 8 days of age had distorted nuclei with condensed matrix and abundant secretory granules associated with rough endoplasmic reticulum and Golgi apparatus. At 8 days of age, an accumulation of fingerlike cytoplasmic processes that extended into the separated intercellular spaces and along the basal aspects of the endometrial epithelial cells were also observed. At 5-8 days of age, the proliferative activity of endometrial epithelial cells in DES-treated mice, identified by bromodeoxyuridine labeling, was significantly lower (10.5-1.7%) than that of the controls (25.5-19.8%). In situ analysis of endometrial luminal epithelial cells for DNA fragmentation representing apoptosis revealed < or = 0.1% and > 10% in the DES-treated and control mice at 5-8 days of age, respectively. The data show that cell cycle kinetics, in addition to changes in morphology, are altered in the developing mouse uterus following neonatal exposure to DES. (+info)
Analysis of mice carrying targeted mutations of the glucocorticoid receptor gene argues against an essential role of glucocorticoid signalling for generating adrenal chromaffin cells.
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Molecular mechanisms underlying the generation of distinct cell phenotypes is a key issue in developmental biology. A major paradigm of determination of neural cell fate concerns the development of sympathetic neurones and neuroendocrine chromaffin cells from a common sympathoadrenal (SA) progenitor cell. Two decades of in vitro experiments have suggested an essential role of glucocorticoid receptor (GR)-mediated signalling in generating chromaffin cells. Targeted mutation of the GR should consequently abolish chromaffin cells. The present analysis of mice lacking GR gene product demonstrates that animals have normal numbers of adrenal chromaffin cells. Moreover, there are no differences in terms of apoptosis and proliferation or in expression of several markers (e.g. GAP43, acetylcholinesterase, adhesion molecule L1) of chromaffin cells in GR-deficient and wild-type mice. However, GR mutant mice lack the adrenaline-synthesizing enzyme PNMT and secretogranin II. Chromaffin cells of GR-deficient mice exhibit the typical ultrastructural features of this cell phenotype, including the large chromaffin granules that distinguish them from sympathetic neurones. Peripherin, an intermediate filament of sympathetic neurones, is undetectable in chromaffin cells of GR mutants. Finally, when stimulated with nerve growth factor in vitro, identical proportions of chromaffin cells from GR-deficient and wild-type mice extend neuritic processes. We conclude that important phenotypic features of chromaffin cells that distinguish them from sympathetic neurones develop normally in the absence of GR-mediated signalling. Most importantly, chromaffin cells in GR-deficient mice do not convert to a neuronal phenotype. These data strongly suggest that the dogma of an essential role of glucocorticoid signalling for the development of chromaffin cells must be abandoned. (+info)
Effects of the neurotrophin brain-derived neurotrophic factor in an experimental model of retinal detachment.
(80/5854)
PURPOSE: To examine the effects of brain-derived neurotrophic factor (BDNF) in an animal model of retinal detachment. METHODS: Cat retinas were detached from the retinal pigment epithelium for either 7 or 28 days. Animals received either an intravitreal injection of BDNF (100 ILg) or phosphate-buffered saline (PBS), the vehicle for BDNF. Retinas were evaluated using morphology and immunocytochemistry. The width of the outer segment zone was measured, and the retinas were evaluated for changes in protein expression by labeling with antibodies to rod opsin, phosducin, synaptophysin, calbindin D, and glial fibrillary acidic protein (GFAP). The effect of BDNF on both proliferation and apoptotic cell death was examined. RESULTS: Although there was variability in the treated retinas, most of the animals receiving BDNF had well-organized outer segments that were longer than those in vehicle-treated controls. Immunocytochemistry revealed that treated retinas had consistently less opsin redistribution to the plasma membrane, less phosducin upregulation, and fewer calbindin D-labeled horizontal cell processes. BDNF did not reduce overall cell death in the detachments or death of photoreceptors by apoptosis. However, it significantly reduced the proliferative response of Miller cells and the extent of upregulation of GFAP. CONCLUSIONS. The results suggest that BDNF may aid in the recovery of the retina after reattachment by maintaining the surviving photoreceptor cells, by reducing the gliotic effects in Muller cells, and perhaps by promoting outer segment regeneration. (+info)