This paper aimed to investigate the role and potential mechanism of p53 on primordial follicle activation. Firstly, the p53 mRNA expression in the ovary of neonatal mice at 3, 5, 7 and 9 days post-partum (dpp) and the subcellular localization of p53 were detected to confirm the expression pattern of p53. Secondly, 2 dpp and 3 dpp ovaries were cultured with p53 inhibitor Pifithrin-μ (PFT-μ, 5 μmol/L) or equal volume of dimethyl sulfoxide for 3 days. The function of p53 in primordial follicle activation was determined by hematoxylin staining and whole ovary follicle counting. The proliferation of cell was detected by immunohistochemistry. The relative mRNA levels and protein levels of the key molecules involved in the classical pathways associated with the growing follicles were examined by immunofluorescence staining, Western blot and real-time PCR, respectively. Finally, rapamycin (RAP) was used to intervene the mTOR signaling pathway, and ovaries were divided into four groups: Control, RAP (1 μmol/L), PFT-μ (5 μmol/L), PFT-μ (5 μmol/L) + RAP (1 μmol/L) groups. The number of follicles in each group was determined by hematoxylin staining and whole ovary follicle counting. The results showed that the expression of p53 mRNA was decreased with the activation of primordial follicles in physiological condition. p53 was expressed in granulosa cells and oocyte cytoplasm of the primordial follicles and growing follicles, and the expression of p53 in the primordial follicles was higher than that in the growing follicles. Inhibition of p53 promoted follicle activation and reduced the primordial follicle reserve. Inhibition of p53 promoted the proliferation of the granulosa cells and oocytes. The mRNA and protein expression levels of key molecules in the PI3K/AKT signaling pathway including AKT, PTEN, and FOXO3a were not significantly changed after PFT-μ treatment, while the expression of RPS6/p-RPS6, the downstream effectors of the mTOR signaling pathway, was upregulated. Inhibition of both p53 and mTOR blocked p53 inhibition-induced primordial follicle activation. Collectively, these findings suggest that p53 may inhibit primordial follicle activation through the mTOR signaling pathway to maintain the primordial follicle reserve.
Female ; Animals ; Mice ; Tumor Suppressor Protein p53/metabolism* ; Proto-Oncogene Proteins c-akt/metabolism* ; Phosphatidylinositol 3-Kinases/metabolism* ; Hematoxylin ; Signal Transduction/physiology* ; TOR Serine-Threonine Kinases ; Sirolimus ; RNA, Messenger

OBJECTIVETo observe the effect of Angelica sinensis polysaccharides (ASP) on the length of telomere, the activity of telomerase and the expression of P53 protein in mice hematopoietic stem cells (HSCs), and explore ASP's potential mechanism for regulating HSC aging.

METHODC57BL/6J mice were randomly divided into the normal group, the aging group and the intervention group. The aging group was radiated with X ray to establish the mice aging HSC model. The intervention group was orally administered with ASP during X-ray irradiation, while the normal group was orally administered with NS. Their HSCs were isolated by immunomagnetic beads. Cell cycles analysis and senescence-associated beta-galactosidase (SA-beta-Gal) staining were used to detect changes in aging HSCs. The expression of P53 was determined by western blot analysis. The length of telomere and the vitality of telomerase were analyzed by southern blot and TRAP-PCR, respectively.

RESULTCompared with the normal group, X-ray irradiation could significantly increase the cell ratio of in HSC G1 stage, rate of SA-beta-Gal positive cells and expression of P53 protein, and reduce the length of telomere and the vitality of telomerase. Compared with the aging group, ASP could significantly inhibit the cell ratio of in HSC G1 stage and the increase in the number of SA-beta-Gal positive cells, down-regulate the expression of P53 protein, and increase the length of telomere and the vitality of telomerase in HSCs.

CONCLUSIONASP could antagonize X-ray-induced aging of HSCs, which may be related to the increase in the length of telomere and the activity of telomerase, as well as the down-regulation of the expression of P53 protein.

Angelica sinensis ; chemistry ; Animals ; Cell Cycle ; drug effects ; physiology ; Cellular Senescence ; drug effects ; physiology ; Female ; Hematopoietic Stem Cells ; cytology ; drug effects ; metabolism ; Male ; Mice ; Mice, Inbred C57BL ; Plant Extracts ; chemistry ; pharmacology ; Plants, Medicinal ; chemistry ; Polysaccharides ; isolation & purification ; pharmacology ; Telomerase ; biosynthesis ; metabolism ; Telomere ; drug effects ; metabolism ; Tumor Suppressor Protein p53 ; biosynthesis ; metabolism

OBJECTIVETo investigate the effect of Notch1 on cell cycle, apoptosis and migration of laryngeal squamous cell carcinoma cell line Hep-2 and explore its possible molecular mechanism.

METHODSHep-2 cells were divided into three groups: untreated group, empty vector group and Notch1 group. The effects of upregulation of Notch1 expression on cell proliferation, cell cycle and apoptosis were assessed by CCK-8 staining and flow cytometry, respectively, and effect of upregulation of Notch1 expression on cell migration of Hep-2 cells was studied using Boyden chamber assay, and further expression changes of genes related to cell proliferation, cell cycle, apoptosis and migration were detected by semi-quantitative RT-PCR and Western blotting.

RESULTSCompared with the untreated group and empty vector group, cell proliferation of Hep-2 in the Notch1 group was significantly inhibited (P < 0.05). The results of flow cytometry revealed that the percentage of cells at G0/G1 phase in the Notch1 group was (70.43 +/- 1.36)%, significantly higher than the (46.39 +/- 1.19)% in the untreated group or (48.41 +/- 1.18)% in the empty vector group, and there was a significant difference among the three groups (P = 0.000). In addition, the percentage of apoptotic cells in the Notch1 group was (22.46 +/- 0.90)%, significantly higher than that in the untreated group [(5.77 +/- 0.37)%] or empty vector group [(6.09 +/- 0.20)%], with a significant difference among the three groups (P = 0.000). The results of Boyden chamber assay demonstrated that the number of cells migrated through membrane in the Notch1 group was evidently lower than that in the untreated group and empty vector group. Moreover, the results of semi-quantitative RT-PCR and Western blotting showed that cell proliferation inhibition and cell cycle arrest were closely associated with downregulation of cyclin D1, cyclin E and CDK2 expressions and upregulation of p53 expression. In addition, upregulation of Notch1 expression mediated cell apoptosis was tightly related to upregulation of caspase 3/9 expressions and downregulation of Bcl-2, while the decrease of Hep-2 cell migration ability was obviously correlated with downregulation of MMP-2/-9 expressions.

CONCLUSIONSNotch1 signaling pathway may play a pivotal role in the occurrence and development of laryngeal squamous cell carcinoma. Further study may elucidate that Notchl signaling pathway may become a molecular target for therapy of laryngeal squamous cell carcinoma.

Apoptosis ; Carcinoma, Squamous Cell ; metabolism ; pathology ; Caspase 3 ; metabolism ; Caspase 9 ; metabolism ; Cell Cycle ; Cell Line, Tumor ; Cell Movement ; Cell Proliferation ; Cyclin D1 ; metabolism ; Cyclin E ; metabolism ; Cyclin-Dependent Kinase 2 ; metabolism ; Humans ; Laryngeal Neoplasms ; metabolism ; pathology ; Matrix Metalloproteinase 2 ; metabolism ; Matrix Metalloproteinase 9 ; metabolism ; Oncogene Proteins ; metabolism ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Receptor, Notch1 ; metabolism ; physiology ; Signal Transduction ; Tumor Suppressor Protein p53 ; metabolism

Animals ; Antineoplastic Agents ; therapeutic use ; Apoptosis ; Apoptosis Regulatory Proteins ; metabolism ; Autophagy ; physiology ; Beclin-1 ; Humans ; Membrane Proteins ; metabolism ; Neoplasms ; drug therapy ; metabolism ; pathology ; Signal Transduction ; TOR Serine-Threonine Kinases ; metabolism ; Tumor Suppressor Protein p53 ; metabolism

Silent information regulator factor 2-related enzyme 1 (Sirtuins 1, SIRT1) is a nicotinamide adenine dinucleotide (NAD)-dependent deacetylase, which can deacetylate histone and non-histone proteins and other transcription factors, and is involved in the regulation of many physiological functions, including gene transcription, energy metabolism, cell senescence and oxidative stress. Recent studies show that through adjusting the activity of endothelial nitric oxide syntheses (eNOS), p53, forkhead box class O (FOXO) and nuclear factor kappa B (NF-kappaB), SIRT1 can protect the functions of vascular endothelia and nerves in a variety of pathological conditions. Therefore, SIRT1 may be used as a potential therapeutic target of these diseases, particularly erectile dysfunction, which are associated with endothelial dysfunction.
Endothelium, Vascular ; physiology ; Erectile Dysfunction ; Forkhead Box Protein O1 ; Forkhead Transcription Factors ; metabolism ; Humans ; Male ; NAD ; metabolism ; NF-kappa B ; metabolism ; Nitric Oxide Synthase Type III ; metabolism ; Oxidative Stress ; Sirtuin 1 ; physiology ; Tumor Suppressor Protein p53 ; metabolism

It has been proposed that the pro-inflammatory catalytic activity of cyclooxygenase-2 (COX-2) plays a key role in the aging process. However, it remains unclear whether the COX-2 activity is a causal factor for aging and whether COX-2 inhibitors could prevent aging. We here examined the effect of COX-2 inhibitors on aging in the intrinsic skin aging model of hairless mice. We observed that among two selective COX-2 inhibitors and one non-selective COX inhibitor studied, only NS-398 inhibited skin aging, while celecoxib and aspirin accelerated skin aging. In addition, NS-398 reduced the expression of p53 and p16, whereas celecoxib and aspirin enhanced their expression. We also found that the aging-modulating effect of the inhibitors is closely associated with the expression of type I procollagen and caveolin-1. These results suggest that pro-inflammatory catalytic activity of COX-2 is not a causal factor for aging at least in skin and that COX-2 inhibitors might modulate skin aging by regulating the expression of type I procollagen and caveolin-1.
Animals ; Aspirin/administration & dosage ; Catalysis ; Caveolin 1/genetics/metabolism ; Collagen Type I/genetics/metabolism ; *Cyclooxygenase 2/metabolism/physiology ; Cyclooxygenase 2 Inhibitors/*administration & dosage ; Gene Expression Regulation ; Mice ; Nitrobenzenes/*administration & dosage ; Pyrazoles/administration & dosage ; Skin Aging/*drug effects/physiology ; Sulfonamides/*administration & dosage ; Tumor Suppressor Protein p53/genetics/metabolism

Curcumin, an active ingredient of dietary spice used in curry, has been shown to exhibit anti-oxidant, anti-inflammatory and anti-proliferative properties. Using EB directed differentiation protocol of H-9 human embryonic stem (ES) cells; we evaluated the effect of curcumin (0-20 μmol/L) in enhancing such differentiation. Our results using real time PCR, western blotting and immunostaining demonstrated that curcumin significantly increased the gene expression and protein levels of cardiac specific transcription factor NKx2.5, cardiac troponin I, myosin heavy chain, and endothelial nitric oxide synthase during ES cell differentiation. Furthermore, an NO donor enhanced the curcumin-mediated induction of NKx2.5 and other cardiac specific proteins. Incubation of cells with curcumin led to a dose dependent increase in intracellular nitrite to the same extent as giving an authentic NO donor. Functional assay for second messenger(s) cyclic AMP (cAMP) and cyclic GMP (cGMP) revealed that continuous presence of curcumin in differentiated cells induced a decrease in the baseline levels of cAMP but it significantly elevated baseline contents of cGMP. Curcumin addition to a cell free assay significantly suppressed cAMP and cGMP degradation in the extracts while long term treatment of intact cells with curcumin increased the rates of cAMP and cGMP degradation suggesting that this might be due to direct suppression of some cyclic nucleotide-degrading enzyme (phosphodiesterase) by curcumin. These studies demonstrate that polyphenol curcumin may be involved in differentiation of ES cells partly due to manipulation of nitric oxide signaling.
Animals ; Antioxidants ; pharmacology ; Cell Differentiation ; drug effects ; Cells, Cultured ; Curcumin ; pharmacology ; Cyclic GMP ; metabolism ; Embryoid Bodies ; drug effects ; metabolism ; physiology ; Enzyme Activators ; pharmacology ; Gene Expression ; drug effects ; Guanylate Cyclase ; genetics ; metabolism ; Homeobox Protein Nkx-2.5 ; Homeodomain Proteins ; genetics ; metabolism ; Humans ; Mice ; Myosin Heavy Chains ; genetics ; metabolism ; Nitric Oxide ; metabolism ; Nitric Oxide Donors ; pharmacology ; Nitric Oxide Synthase Type III ; genetics ; metabolism ; Nitroso Compounds ; pharmacology ; Pyrazoles ; pharmacology ; Pyridines ; pharmacology ; Second Messenger Systems ; Transcription Factors ; genetics ; metabolism ; Troponin ; genetics ; metabolism ; Tumor Suppressor Protein p53 ; metabolism

PURPOSE: The purpose of this study is to understand the mechanism of apoptosis occurring on a cultured human lens epithelial cell line after exposure to ultraviolet (UV) light. We intended to confirm the presence of cellular toxicity and apoptosis and to reveal the roles of p53, caspase 3 and NOXA in these processes. METHODS: Cells were irradiated with an ultraviolet lamp. Cellular toxicity was measured by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Hoechst staining and fluorescent anti-caspase 3 antibodies were used for apoptosis investigation. The quantities of p53, caspase 3, and NOXA were measured by Western blotting for to investigate the apoptosis pathway. RESULTS: Cellular toxicity on the human lens epithelium markedly increased with time after UV exposure. On Hoechst staining, we found that apoptosis also remarkably increased after exposure to ultraviolet light, compared with a control group. In the immunochemical study using anti-caspase 3 antibodies, active caspase 3 significantly increased after exposure to ultraviolet light. On Western blotting, p53 decreased, while caspase 3 and NOXA increased. CONCLUSIONS: Exposure of cultured human lens epithelial cell lines to ultraviolet light induces apoptosis, which promotes the expression of NOXA and caspase 3 increases without increasing p53. This may suggest that UV induced apoptosis is caused by a p53-independent pathway in human lens epithelial cells.
Apoptosis/*physiology ; Caspase 3/metabolism ; Cell Line ; Cell Survival/radiation effects ; Epithelial Cells/radiation effects ; Humans ; Lens, Crystalline/cytology/*physiology/*radiation effects ; Proto-Oncogene Proteins c-bcl-2/metabolism ; Tumor Suppressor Protein p53/metabolism ; *Ultraviolet Rays

OBJECTIVETo investigate the role of Wnt/β-catenin signaling in aging of mesenchymal stem cells (MSCs) of rats.

METHODSSerum samples were collected from young (8 ≈ 12 w) and aged (64 ≈ 72 w) SD rat. Four experiment groups were assigned: young rat serum (YRS), YRS+Wnt 3a, old rat serum (ORS) and ORS+DKK1 groups. Immunofluorescence and Western blotting were used to detect the expression of intracellular β-catenin. The senescence-associated changes were examined with SA-β-galactosidase staining. The proliferation ability was tested by MTT assays. The survived and apoptotic cells were determined by AO/EB staining. The expressions of γ-H2A. X and p53 protein were detected by immunofluorescence and Western blotting. RT-PCR was used to detect the expression of p53 and p21 mRNA.

RESULTSCompared with the YRS group, the intracellular expression of β-catenin in the ORS group was significantly increased,especially in the nuclei of MSCs. After treatment of DKK1 in ORS, the γ-catenin expression was reduced. The number of SA-β-galactosidase positive MSCs was significantly higher in the YRS+Wnt 3a group than that in the YRS group (P<0.01), and the proliferative and survival ability of MSCs was significantly decreased in the YRS+Wnt 3a group. The number of SA-β-galactosidase positive MSCs in the ORS+DKK1 group was significantly decreased compared with that in ORS group (P <0.01), and the proliferative and survival ability of MSCs was significantly increased in the ORS+DKK1 group. The expression of γ-H2A.X, p53 and p21 was markedly increased in the ORS group than that in YRS group, however, after treatment with Wnt/β-catenin signaling inhibitor DKK1, the expression of γ-H2A.X, p53 and p21 was significantly decreased compared with that in the ORS group.

CONCLUSIONResults suggest that the Wnt/β-catenin signaling is activated in the MSCs cultured with ORS and excessive activation of Wnt/β-catenin signaling can promote MSCs aging. The DNA damage response and p53/p21 pathway may be main mediators of MSC aging induced by excessive activation of Wnt/β-catenin signaling.

Animals ; Cell Proliferation ; Cell Survival ; physiology ; Cells, Cultured ; Cellular Senescence ; physiology ; Mesenchymal Stromal Cells ; metabolism ; physiology ; Rats ; Rats, Sprague-Dawley ; Signal Transduction ; Tumor Suppressor Protein p53 ; metabolism ; Wnt Proteins ; metabolism ; beta Catenin ; metabolism

Antineoplastic Agents, Alkylating ; pharmacology ; Apoptosis ; drug effects ; Benzimidazoles ; pharmacology ; DNA Repair ; Dacarbazine ; analogs & derivatives ; pharmacology ; Drug Resistance, Neoplasm ; drug effects ; physiology ; Humans ; Membrane Proteins ; metabolism ; O(6)-Methylguanine-DNA Methyltransferase ; antagonists & inhibitors ; metabolism ; Poly(ADP-ribose) Polymerase Inhibitors ; Poly(ADP-ribose) Polymerases ; metabolism ; Proto-Oncogene Proteins ; metabolism ; Purines ; pharmacology ; Pyrimidines ; pharmacology ; Tumor Suppressor Protein p53 ; metabolism