Many extracellular matrix molecules are expressed in the embryonic nervous system and there is some evidence that they are important regulators of neural development. Of these molecules, laminin appears to be the most potent, affecting virtually all neurons of the peripheral and central nervous system. This study was undertaken to investigate the effects of laminin on the proliferation and differentiation of cultured neuroepithelial cells taken from fetal rat forebrains (embryonic day 17-19). The results are summarized as follows. 1) Neuroepithelial cells cultivated in epidermal growth factors containing serum-free medium subsequently differentiated into neurons, astrocytes, and oligodendrocytes. 2) Neuronal cells derived from neuroepithelial cells were immunoreactive for gamma-aminobutyric acid (GABA) or substance P, but were not for serotonin and tyrosine hydroxylase. 3) In western blot analysis, the phosphorylated neurofilament content in neuronal cells was higher in culture on laminin than in culture on poly-L-lysine (PLL). 4) The proliferation rate of GABAergic neurons was higher in culture on laminin than in culture on PLL. These results suggest that GABAergic and substance P-ergic neurons can be differentiated from neuroepithelial cells and that laminin promotes the differentiation of neuronal cells from neuroepithelial cells and the increased proliferation rate of GABAergic cells.
Animal ; Brain/drug effects* ; Brain/cytology ; Cell Aging/drug effects ; Cell Differentiation/drug effects ; Cell Division/drug effects ; Cells, Cultured ; Epidermal Growth Factor/pharmacology* ; Epithelial Cells/drug effects ; Epithelial Cells/cytology ; GABA/physiology ; Laminin/pharmacology* ; Neurons/physiology ; Neurons/drug effects* ; Neurons/cytology* ; Rats/embryology

Hydroxyurea is commonly used to treat hematologic disorders and some type of solid tumors, but the mechanism for its therapeutic effect is not clearly known. In this study, we examined the effect of hydroxyurea on rat hepatoma McA-RH7777 cells, specifically, on the role of mitogen-activated protein (MAP) kinase signal transduction pathways and p21Waf1, p27Kip1 and p53. Rat hepatoma McA-RH7777 cells treated with hydroxyurea for 7 days, caused the inhibition of cell growth in a dose-dependent manner. But, this growth inhibition was not caused by necrosis or apoptosis but instead was associated with cell senescence-like change as evidenced by senescence associated-beta-galactosidase staining, and cells arrest at G1 phase of cell cycle. Phosphorylation of MAP kinases, such as ERK, JNK, and p38, was found to be decreased after treatment of cells with hydroxyurea. But, the expression of p21Waf1 was increased, while p27Kip1 and p53 were not detected in hydroxyurea treated rat hepatoma cells. Hydroxyurea treatment induced G1 arrest and a senescence-like changes in rat hepatoma McA-RH7777 cells may be the likely results of signal disruption of MAP kinases (ERK, JNK, and p38 MAP kinase) and p21Waf1 over-expression.
Animals ; Antineoplastic Agents/*pharmacology ; Cell Aging/drug effects ; Cell Cycle Proteins/analysis/metabolism/*physiology ; Cell Line, Tumor ; Cell Proliferation/drug effects ; G1 Phase/drug effects/physiology ; Hydroxyurea/*pharmacology ; Liver Neoplasms, Experimental/enzymology/*metabolism ; Mitogen-Activated Protein Kinases/analysis/*physiology ; Phosphorylation/drug effects ; Protein p53/analysis/metabolism ; Rats ; Research Support, Non-U.S. Gov't ; Tumor Suppressor Proteins/analysis/metabolism ; Up-Regulation

PURPOSE: To investigate the effect of advanced glycation end products (AGE) on oxidative stress and cellular senescence in cultured human trabecular meshwork cells (HTMC). METHODS: Primarily cultured HTMC were exposed to 0, 10, 50, 100, 200 microg/mL of glycated bovine serum albumin (G-BSA) for 5 days. Also co-exposed were L-arginine, sepiapterin, and antioxidant N-acetylcysteine (NAC). Cellular survival and production of nitric oxide (NO), superoxide, and reactive oxygen species were assessed by 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide assay, Griess assay, cytochrome c assay, and dichlorofluorescin diacetate assay, respectively. Senescence-associated beta-galactosidase staining was performed to quantify the degree of cellular senescence. RESULTS: G-BSA decreased cellular survival, NO production, and increased superoxide production significantly in a dose-dependent manner. The effects of G-BSA were abolished with co-exposure of L-arginine, sepiapterin, and NAC. G-BSA enhanced cellular senescence accompanied by increased production of reactive oxygen species. G-BSA-induced cellular senescence was suppressed by application of L-arginine, sepiapterin, and NAC. CONCLUSIONS: AGE enhances cellular senescence of HTMC accompanied with increased oxidative stress. AGE-induced oxidative stress and cellular senescence could be delayed by application of anti-oxidants.
Acetylcysteine/metabolism ; Apoptosis/drug effects/physiology ; Arginine/metabolism ; Cell Aging/drug effects/*physiology ; Cell Survival/drug effects/physiology ; Cells, Cultured ; Glycosylation End Products, Advanced/metabolism/*toxicity ; Humans ; Nitric Oxide/metabolism ; Oxidative Stress/*physiology ; Pterins/metabolism ; Reactive Oxygen Species/metabolism ; Serum Albumin, Bovine/metabolism/toxicity ; Trabecular Meshwork/drug effects/*metabolism/*pathology

PURPOSE: Foxo3 in female reproduction has been reported to regulate proliferation of granulose cells that form follicles. There are no reports so far that discuss on the role of Foxo3 in males. This study was designed to outline the role of Foxo3 in the testes. MATERIALS AND METHODS: Testes from mice at birth to postpartum week (PPW) 5 were isolated and examined for the expression of Foxo3 using immunostaining. To elucidate role of Foxo3 in Leydig cells, R2C cells were treated with luteinizing hormone (LH) and the phosphorylation of Foxo3. Testosterone and steroidogenic acute regulatory (StAR) protein levels were measured after constitutive active [triple mutant (TM)] human FOXO3 adenovirus was transduced and StAR promoter assay was performed. RESULTS: Foxo3 expression in the testicles started from birth and lasted until PPW 3. After PPW 3, most Foxo3 expression occurred in the nuclei of Leydig cells; however, at PPW 5, Foxo3 was expressed in both the nucleus and cytoplasm. When R2C cells were treated with luteinizing hormone, Foxo3 phosphorylation levels by AKT increased. After blocking the PI3K pathway, LH-induced phosphorylated Foxo3 levels decreased, indicating that LH signaling regulates Foxo3 localization. When active FOXO3-TM adenovirus was introduced into a Leydig tumor cell line, the concentrations of testosterone and StAR protein decreased. When FOXO3 and a StAR promoter vector were co-transfected into HEK293 cells for a reporter assay, FOXO3 inhibited the StAR promoter. CONCLUSION: FOXO3 affects testosterone synthesis by inhibiting the formation of StAR protein. LH hormone, meanwhile, influences Foxo3 localization, mediating its function.
Animals ; Cell Aging/*physiology ; Cell Nucleus/metabolism ; Cytoplasm/metabolism ; Forkhead Transcription Factors/*metabolism ; HEK293 Cells ; Humans ; Leydig Cells/*drug effects/*enzymology/metabolism ; Luteinizing Hormone/blood ; Male ; Mice ; Phosphatidylinositol 3-Kinases ; Phosphoproteins/metabolism ; Phosphorylation ; Signal Transduction/drug effects ; Testosterone/blood/*metabolism

AIMTo investigate the effect of cocaine on apoptosis and caspase-3 activity in germ cells in male rats at different ages.

METHODSCocaine hydrochloride was given (15 mg/kg body weight s.c.) to male Sprague-Dawley rats of 3 weeks (n = 8), 6 weeks (n = 8) and 12 weeks (n = 8) of age, daily for 28 Days. The serum levels of follicle stimulating hormone (FSH), luteinizing hormone (LH), prolactin (PRL), testosterone (T) and estrogen (E2) were assayed, and the DNA fragmentation of germ cells was determined by gel eletronphoresis. The cell cycle, apoptosis and caspase-3 activity of germ cells were tested by flow cytometry.

RESULTSAfter the 28-day cocaine treatment, testes weight of the 3-week-old rats, the testes and body weights of the 6-week-old rats were decreased significantly compared to those of their corresponding controls (P < 0.05). The serum level of T was decreased significantly in the 3-week-old and 6-week-old rats, and the serum level of PRL was also decreased significantly in 12-week-old rats compared to the controls (P < 0.05). In all the three cocaine-treated groups, the isolated DNA displayed a clear ladder pattern, especially in the 6-week old rats. The number of apoptosic germ cells increased significantly in 3- and 6-week-old rats treated with cocaine (P < 0.05). The caspase-3 activity in all three groups increased significantly compared to the controls (P < 0.05), especially in the 6-week-old rats.

CONCLUSIONCocaine exposure for 28 Days leads to significant damage to male gonad and apoptosis elevation in testes of rats of different ages, especially in those of 6 weeks of age. The increase in caspase-3 activity might be a key pathway related to the early stage of apoptosis as the mechanism of cocaine-induced germ cell loss.

Aging ; physiology ; Animals ; Caspase 3 ; Caspases ; drug effects ; metabolism ; Cell Cycle ; drug effects ; Cocaine ; pharmacology ; Estrogens ; blood ; Follicle Stimulating Hormone ; blood ; Luteinizing Hormone ; blood ; Male ; Prolactin ; blood ; Rats ; Rats, Sprague-Dawley ; Spermatozoa ; cytology ; drug effects ; physiology ; Testis ; drug effects ; pathology ; Testosterone ; blood

Adipose tissue is an important endocrine regulator of glucose metabolism and energy homeostasis. Researches have focused on this tissue not only as a target for pharmacotherapy of obesity and insulin resistance but also as an endocrine tissue with leptin secretion and high insulin sensitivity. Brown adipose tissue (BAT) additionally plays a unique role in thermoregulation through the mitochondrial uncoupling protein 1 (UCP1), which uncouples oxidative phosphorylation. As a genetic tissue ablation model of BAT, we made transgenic mice expressing herpes simplex virus thymidine kinase (HSV-TK) driven by the brown adipocyte- specific UCP1 minimal regulatory element. The HSV-TK transgene was expressed specifically in BAT and more than 35% increase of apoptosis was induced by ganciclovir (GCV) treatment. Nevertheless, the expression level was not high enough to induce BAT ablation in GCV-treated adult mice. Importantly, however, we found that brown adipocytes in the periphery of interscapular BAT were transformed into white adipocyte-like unilocular cells. These cells express white adipocyte-specific leptin protein but are different in the ultrastructure of mitochondria from classical white adipocytes. Our data indicates that atrophy of BAT causes transformation into white adipocyte-like cells in the adult mouse and also suggests that further molecular understanding of adipocyte plasticity using our transgenic mouse model might be beneficial for the development of anti-obesity/anti-diabetic therapies.
Adipose Tissue/*cytology/drug effects/metabolism/ultrastructure ; Aging/physiology ; Animals ; Body Weight ; Carrier Proteins/genetics/metabolism ; *Cell Differentiation/drug effects ; Ganciclovir/pharmacology ; Ion Channels ; Leptin/metabolism ; Membrane Proteins/genetics/metabolism ; Mice ; Mice, Transgenic ; Mitochondrial Proteins ; Obesity/chemically induced ; Organ Specificity ; Thymidine Kinase/genetics/metabolism

Lysophosphatidic acid (LPA) is a phospholipid growth factor that acts through G-protein-coupled receptors. Previously, we demonstrated an altered profile of LPA-dependent cAMP content during the aging process of human diploid fibroblasts (HDFs). In attempts to define the molecular events associated with the age-dependent changes in cAMP profiles, we determined the protein kinase A (PKA) activity, phosphorylation of cAMP-response element binding protein (CREB), and the protein expression of CRE-regulatory genes, c-fos and COX-2 in young and senescent HDFs. We observed in senescent cells, an increase in mRNA levels of the catalytic subunit a of PKA and of the major regulatory subunit Ia. Senescence-associated increase of cAMP after LPA treatment correlated well with increased CREB phosphorylation accompanying activation of PKA in senescent cells. In senescent cells, after LPA treatment, the expression of c-fos and COX-2 decreased initially, followed by an increase. In young HDFs, CREB phosphorylation decreased following LPA treatment, and both c-fos and COX-2 protein levels increased rapidly. CRE-luciferase assay revealed higher basal CRE-dependent gene expression in young HDFs compared to senescent HDFs. However, LPA-dependent slope of luciferase increased more rapidly in senescent cells than in young cells, presumably due to an increase of LPA-induced CREB phosphorylation. CRE-dependent luciferase activation was abrogated in the presence of inhibitors of PKC, MEK1, p38MAPK, and PKA, in both young and senescent HDFs. We conclude that these kinase are coactivators of the expression of CRE-responsive genes in LPA-induced HDFs and that their changed activities during the aging process contribute to the final expression level of CRE-responsive genes.
Time Factors ; Protein Kinase Inhibitors/pharmacology ; Phosphorylation ; Male ; Lysophospholipids/*pharmacology ; Luciferases/genetics/metabolism ; Humans ; Gene Expression/drug effects ; Fibroblasts/cytology/*drug effects/metabolism ; Diploidy ; Cyclic AMP-Dependent Protein Kinases/genetics/metabolism ; Cyclic AMP Response Element-Binding Protein/metabolism ; Cyclic AMP/*metabolism ; Cells, Cultured ; Cell Aging/physiology ; Catalytic Domain/genetics

Alzheimer's disease (AD) is the most common cause of age-related dementia. The neuropathological hallmarks of AD include extracellular deposition of amyloid-beta peptides and neurofibrillary tangles that lead to intracellular hyperphosphorylated tau in the brain. Soluble amyloid-beta oligomers are the primary pathogenic factor leading to cognitive impairment in AD. Neural stem cells (NSCs) are able to self-renew and give rise to multiple neural cell lineages in both developing and adult central nervous systems. To explore the relationship between AD-related pathology and the behaviors of NSCs that enable neuroregeneration, a number of studies have used animal and in vitro models to investigate the role of amyloid-beta on NSCs derived from various brain regions at different developmental stages. However, the Abeta effects on NSCs remain poorly understood because of conflicting results. To investigate the effects of amyloid-beta oligomers on human NSCs, we established amyloid precursor protein Swedish mutant-expressing cells and identified cell-derived amyloid-beta oligomers in the culture media. Human NSCs were isolated from an aborted fetal telencephalon at 13 weeks of gestation and expanded in culture as neurospheres. Human NSCs exposure to cell-derived amyloid-beta oligomers decreased dividing potential resulting from senescence through telomere attrition, impaired neurogenesis and promoted gliogenesis, and attenuated mobility. These amyloid-beta oligomers modulated the proliferation, differentiation and migration patterns of human NSCs via a glycogen synthase kinase-3beta-mediated signaling pathway. These findings contribute to the development of human NSC-based therapy for AD by elucidating the effects of Abeta oligomers on human NSCs.
Amyloid beta-Peptides/*pharmacology ; Animals ; Apoptosis ; Cell Aging ; Cell Movement ; Cell Proliferation ; Culture Media, Conditioned/chemistry/pharmacology ; Fetus/cytology ; Glycogen Synthase Kinase 3/*metabolism ; HEK293 Cells ; Humans ; Mice ; Mice, Inbred C57BL ; Neural Stem Cells/*drug effects/metabolism/physiology ; Signal Transduction ; Telomere Shortening