1.Posttranscriptional induction of p21Waf1 mediated by ectopic p16INK4 in human diploid fibroblast.
Xiao-lin HAN ; Fu-guo WU ; Zong-yu ZHANG ; Tan-jun TONG
Chinese Medical Journal 2007;120(5):405-409
BACKGROUNDBoth p16(INK4) and p21(Waf1) are tumor suppressors with similar biological functions in the regulation of cellular senescence. Previous reports showed that p16(INK4) could be activated by p21(Waf1) through transcriptional factor Sp1 in HeLa cells. This study was undertaken to determine the effects of p16(INK4) on the expression and functions of p21(Waf1).
METHODSHuman diploid fibroblast 2BS cells were stably transfected with sense (2BS/p16(INK4)), antisense p16(INK4) (2BS/asp16(INK4)) or empty vector (2BS/neo). Then they were assayed by reverse-transcription polymerase chain reaction (RT-PCR), fluorescence activated cell sorting (FACS) and Western blot.
RESULTS2BS/p16(INK4) cells exhibited cell cycle arrest in both G1 and G2/M phases. Endogenous p21(Waf1) protein levels increased twofold in the 2BS/p16(INK4) cells, but not decreased in the 2BS/asp16(INK4) cells. p21(Waf1) mRNA levels were not affected in neither 2BS/p16(INK4) nor 2BS/asp16(INK4) cells.
CONCLUSIONp16(INK4) may play an important role in the regulation of cellular senescence by modulating the p21(Waf1) protein level via the posttranscriptional mechanism.
Cell Cycle ; Cells, Cultured ; Cellular Senescence ; Cyclin-Dependent Kinase Inhibitor p16 ; physiology ; Cyclin-Dependent Kinase Inhibitor p21 ; physiology ; Fibroblasts ; metabolism ; Humans ; Transcription, Genetic
2.Aberrant Cell Cycle Regulation in Cervical Carcinoma.
Yonsei Medical Journal 2005;46(5):597-613
Carcinoma of the uterine cervix is one of the most common malignancies among women worldwide. Human papillomaviruses (HPV) have been identified as the major etiological factor in cervical carcinogenesis. However, the time lag between HPV infection and the diagnosis of cancer indicates that multiple steps, as well as multiple factors, may be necessary for the development of cervical cancer. The development and progression of cervical carcinoma have been shown to be dependent on various genetic and epigenetic events, especially alterations in the cell cycle checkpoint machinery. In mammalian cells, control of the cell cycle is regulated by the activity of cyclin-dependent kinases (CDKs) and their essential activating coenzymes, the cyclins. Generally, CDKs, cyclins, and CDK inhibitors function within several pathways, including the p16INK4A-cyclin D1-CDK4/6-pRb-E2F, p21WAF1-p27KIP1-cyclinE-CDK2, and p14ARF-MDM2-p53 pathways. The results from several studies showed aberrant regulation of several cell cycle proteins, such as cyclin D, cyclin E, p16 INK4A, p21WAF1, and p27KIP1, as characteristic features of HPV- infected and HPV E6/E7 oncogene-expressing cervical carcinomas and their precursors. These data suggested further that interactions of viral proteins with host cellular proteins, particularly cell cycle proteins, are involved in the activation or repression of cell cycle progression in cervical carcinogenesis.
Uterine Cervical Neoplasms/*pathology
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Tumor Suppressor Protein p53/physiology
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Tumor Suppressor Protein p14ARF/physiology
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Retinoblastoma Protein/physiology
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Proto-Oncogene Proteins c-mdm2/physiology
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Humans
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Female
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E2F Transcription Factors/physiology
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Cyclin-Dependent Kinase Inhibitor p27/physiology
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Cyclin-Dependent Kinase Inhibitor p21/physiology
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Cyclin-Dependent Kinase Inhibitor p16/physiology
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Cyclin-Dependent Kinase 4/physiology
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Cyclin-Dependent Kinase 2/physiology
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Cyclin E/physiology
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Cyclin D1/physiology
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Cell Cycle/*physiology
3.Expression of p16INK4a in mouse endometrium and its effect during blastocyst implantation.
Huan YANG ; Yi XIE ; Rong YANG ; Sha-Li WEI ; Qiang XI
Acta Physiologica Sinica 2008;60(4):547-552
The expression of tumor suppressor gene p16INK4a in mouse endometrium during early pregnancy and its possible role in blastocyst implantation were investigated in the present study. Real-time fluorescent quantitative PCR (FQ-PCR) and immunohistochemistry were applied to detect p16INK4a mRNA and protein expressions in endometrium of un-pregnant and pregnant mice on day 2, 3, 4, 5, 7, respectively. In addition, p16INK4a antibody was injected into the horns of uteri in pregnant mice on day 3 and its effect during blastocyst implantation was detected in vivo. The higher expressions of p16INK4a mRNA and protein were observed in pregnant mice compared with that in un-pregnant mice, with a steady increase from day 2 to day 5 and reaching the maximal level on day 5 of pregnancy and then decreasing. p16INK4a antibody decreased the number of implanted blastocysts compared with that of saline-injected group. The results suggest that p16INK4a may be associated with apoptosis of luminal epithelial cells and decidual cells, coordinating decidualization of endometrium and invasion of trophoblastic cells. Thus, we presume that p16INK4a participates in the process of blastocyst implantation in mice.
Animals
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Blastocyst
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physiology
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Cyclin-Dependent Kinase Inhibitor p16
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physiology
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Embryo Implantation
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Endometrium
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physiology
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Female
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Immunohistochemistry
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Mice
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Pregnancy
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RNA, Messenger
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Real-Time Polymerase Chain Reaction
4.Epstein-Barr virus induces human nasopharyngeal epithelial cells to escape from the replicative senescence.
Jing YANG ; Faqing TANG ; Huanhua GU ; Xiyun DENG ; Xinxian WENG ; Min TANG ; Ya CAO
Chinese Medical Journal 2002;115(6):803-809
OBJECTIVETo observe the biological changes of primary human nasopharyngeal epithelial cells in the early stage of immortalization.
METHODSThe morphological changes of nasopharyngeal epithelial cells were observed by phase contrast microscopy, and the activity profile of senescence-associated beta-galactosidase (SA-beta-Gal) was detected by SA-beta-Gal staining. The expression of p16(INK4a) protein was tested by immunochemical assay, and the life span in vitro of nasopharyngeal epithelial cells was calculated as population doublings. In addition, the expression of Epstein-Barr (EB) virus latent membrane protein 1 (LMP1) was also detected by immunofluorescence staining.
RESULTSMorphologically, cells treated with EB virus and 12-o-tetradecanoyl-phorbol-13-acetate (TPA) formed multi-layer foci, and their cellular life span in vitro was extended (about 155 days of culture). A low percentage of cells (about 4.8%) expressed SA-beta-Gal activity at late primary culture, and did not always express p16(INK4a) protein in the progression of culture.
CONCLUSIONSNasopharyngeal epithelial cells treated with EB virus in cooperation with TPA can pass through the stage of senescence and enter the early stage of immortalization. Some changes of phenotype occur in these cells. Our results provide data for further studying the mechanism of immortalization and the establishment of a human nasopharyngeal epithelial cell line.
Cell Transformation, Viral ; Cellular Senescence ; Cyclin-Dependent Kinase Inhibitor p16 ; analysis ; Epithelial Cells ; physiology ; virology ; Herpesvirus 4, Human ; physiology ; Humans ; Nasopharynx ; cytology ; virology ; Tetradecanoylphorbol Acetate ; pharmacology
5.p16INK4a expression mediated by recombinant adenovirus can induce senescence of A549 cells.
Xue-yuan BAI ; Feng-xiang CHE ; Xiang-mei CHEN ; Ling-ying MENG ; Yu ZHOU
Chinese Journal of Experimental and Clinical Virology 2004;18(1):54-58
OBJECTIVETo construct E1-deletion and replication-defective human type 5 recombinant adenovirus vector and to study the effect of p16INK4a on proliferation and aging of A549 cells.
METHODSp16INK4a cDNA was cloned into pAdCMV to construct recombinant pAdCMV p16INK4a, which was co-transfected into 293 cell together with pJM17. The recombinant p16INK4a adenovirus (Ad-p16INK4a) was generated by homologous recombination and identified with duplex PCR. Lung cancer cell A549, which has a homozygous deletion of p16INK4a gene, was infected with the prepared Ad-p16INK4a virus. X-gal staining and TRAP-ELISA were used for detecting senescence-associated beta-galactosidase and telomerase activities in A549 cells.
RESULTSImmunohistochemical staining and Western blot indicated that p16INK4a gene was transferred into A549 cell with more than 95% efficiency by recombinant adenovirus and p16INK4a protein was expressed at a high level- p16INK4a could markedly inhibit growth of A549 cells, induced expression of senescence-associated beta-galactosidase and suppressed telomerase activity in A549 cells.
CONCLUSIONRecombinant adenovirus vector could efficiently mediate transfer and expression of foreign genes in human cell and could be used for gene immunization and gene therapy; p16INK4a could inhibit A549 cell growth and induce its replicative senescence.
Adenoviridae ; genetics ; Cell Line, Tumor ; Cell Proliferation ; Cellular Senescence ; Cyclin-Dependent Kinase Inhibitor p16 ; biosynthesis ; genetics ; physiology ; Gene Expression ; Genetic Vectors ; Humans ; Recombination, Genetic ; Transfection
6.Effects of arsenic trioxide on cell cycle and expression of cyclin dependent kinase inhibitors of multiple myeloma cells.
Yu-bao CHEN ; Wei-jun FU ; Jian HOU ; Si-qi DING ; Dong-xing WANG ; Zhen-gang YUAN ; Xian-tao KONG
Chinese Journal of Hematology 2003;24(4):193-196
OBJECTIVETo study the effects of arsenic trioxide (As(2)O(3)) on cell cycle and expression of cyclin dependent kinase inhibitors (CDKIs) in multiple myeloma (MM) cells, and explore its pharmacological mechanism.
METHODSThe DNA content of MM cells line HS-Sultan was analyzed by flow cytometry after exposure to As(2)O(3), the effects on expression of CDKI P15, P16 AND P21 were studied by reverse transcriptase PCR.
RESULTSDNA flow cytometric analysis showed that As(2)O(3) induced most of HS-Sultan cells, arrest at G(0)/G(1) phase and a small fraction at G(2)/M phase and apoptosis occurred mainly in S phase. There was no expression of P15 and P16 mRNA in untreated HS-Sultan cells and 1.0 micromol/L As(2)O(3) could make them expressed after exposed 24 or 48 hours respectively. Expression of P12 mRNA was obviously elevated by As(2)O(3) comparing with that of control.
CONCLUSIONOne of the pharmacological mechanisms of As(2)O(3) is to activate the expression of CDKI P15, P16 and P21, and consequently affect cell proliferation cycle.
Antineoplastic Agents ; pharmacology ; Arsenicals ; pharmacology ; Cell Cycle ; drug effects ; physiology ; Cyclin-Dependent Kinase Inhibitor p15 ; biosynthesis ; genetics ; Cyclin-Dependent Kinase Inhibitor p16 ; biosynthesis ; genetics ; Cyclin-Dependent Kinase Inhibitor p21 ; biosynthesis ; genetics ; Humans ; Multiple Myeloma ; drug therapy ; metabolism ; pathology ; Oxides ; pharmacology ; RNA, Messenger ; genetics ; Tumor Cells, Cultured
7.Expression of positive and negative regulators of cell cycle during wound healing.
Xudong ZHU ; Yanfei DI ; Chengxiang HU ; Zhengguo WANG
Chinese Medical Journal 2002;115(3):326-330
OBJECTIVETo detect the expression of cell cycle positive regulators cyclin D(1), cyclin E, CDK(2), CDK(4) and negative regulators p21(cip1), p27(kip1), p16(ink4a) and p15(ink4b) during wound healing in rats.
METHODSOpen wounds of full-thickness skin, diameter 1.8 cm, on rat backs were used as the wound model. Wound tissues were harvested on postwounding days 3, 5, 7, 9, 11, 14, 21 and 30. Ki67 expression in granulation tissue was detected by immunohistochemical assay. The patterns of the expression of cyclin D(1), cyclin E, CDK(2), CDK(4) and p21(cip1), p27(kip1), p16(ink4a), p15(ink4b) were detected by Western blot.
RESULTSCell proliferation in granulation tissue took place predominantly within the first week after injury, with the proliferation peak occurring at postwounding day 5. There were no dramatic variations in the expression of cyclin D(1), CDK(2) and CDK(4) during wound healing. Up-regulated cyclin E was maintained from day 3 to 11 after injury, and then was down-regulated. No expression of p16(ink4a) and p15(ink4b) was found. p21(cip1) was expressed only from day 7 to 14, with peak expression observed on day 9. Constitutive p27(kip1) was expressed throughout wound healing with low levels in the proliferating period of day 3 to 5 and with increased levels in the post-mitotic and remodeling stage. The expression of p21(cip1) and p27(kip1) showed an inverse gradient to that of Ki67.
CONCLUSIONp21(cip1) and p27(kip1) play a supervising role in preventing the hyperproliferative tendency in tissue repair.
Animals ; Cell Cycle ; physiology ; Cell Cycle Proteins ; biosynthesis ; physiology ; Cell Division ; physiology ; Cyclin-Dependent Kinase Inhibitor p16 ; biosynthesis ; Cyclin-Dependent Kinase Inhibitor p27 ; Cyclin-Dependent Kinases ; antagonists & inhibitors ; biosynthesis ; Cyclins ; biosynthesis ; Male ; Rats ; Rats, Wistar ; Skin ; cytology ; metabolism ; Tumor Suppressor Proteins ; biosynthesis ; physiology ; Wound Healing
8.Influence of HPV16 on expression of Rb, p16 and cyclin D1 in oral epithelial cell.
Patima SDEK ; Zhiyuan ZHANG ; Jun CAO
Chinese Journal of Stomatology 2002;37(2):84-86
OBJECTIVETo investigate the role of HPV16E6 and E7 during the transformation of oral epithelial cells.
METHODSAn human immortalized oral epithelial cell line (HIOEC) was established by transfecting HPV16E6, E7 open reading frames using recombinant retroviral system plxsn to human normal oral epithelial cells. Expression of HPV16E6, E7, Rb, P16 and Cycin D1 were analyzed by Western blot in HIOEC and human normal oral epithelial cells. Formation of complex of HPV16E7 and Rb were analyzed by Immunoprecipitation-western blot. Human normal oral epithelial cells and the oral epithelial cells transfected with plxsn were used as control groups.
RESULTSHIOEC expressed HPV16 E6 and E7; HIOEC expressed both hyperphosphorylated and underphosphorylated Rb while oral epithelial cells in two control groups only expressed hyperphosphorylated Rb. HPV16 E7 formed complex with underphosphorylated Rb; the level of P16 and Cyclin D1 had no remarkable change.
CONCLUSIONSHPV16E7 plays an important role in the immortalization of oral epithelial cells induced by HPV16.
Blotting, Western ; Cell Line ; Cell Transformation, Neoplastic ; Cyclin D1 ; analysis ; Cyclin-Dependent Kinase Inhibitor p16 ; analysis ; Humans ; Mouth Mucosa ; metabolism ; pathology ; virology ; Oncogene Proteins, Viral ; physiology ; Papillomavirus E7 Proteins ; Phosphorylation ; Repressor Proteins ; Retinoblastoma Protein ; analysis
9.Physiological testosterone retards cardiomyocyte aging in Tfm mice via androgen receptor-independent pathway.
Li ZHANG ; Da LEI ; Gui-Ping ZHU ; Lei HONG ; Sai-Zhu WU
Chinese Medical Sciences Journal 2013;28(2):88-94
OBJECTIVETo determine whether testosterone modulates markers of cardiomyocytes aging via its classic androgen receptor (AR)-dependent pathway or conversion to estradiol.
METHODSMale littermates and testicular feminized (Tfm) mice were randomly separated into 4 experimental groups littermate controls (n=8), Tfm mice (n=7), testosterone-treated Tfm mice (n=8), and Tfm mice treated with testosterone in combination with the aromatase inhibitor anastrazole (n=7). Cardiomyocytes were isolated from mouse left ventricles, the activity of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), and the amount of malondialdehyde (MDA) were measured using colorimetry method, and expression of p16(INK4α) and retinoblastoma (Rb) proteins were detected by Western blotting.
RESULTSThe SOD and GSH-Px enzyme activities of cardiomyocytes were decreased, and the MDA levels and the expression of p16(INK4α) and Rb proteins were increased in Tfm mice compared with control mice. An increase was observed in the activities of SOD and GSH-Px enzyme as well as a decrease in MDA levels and the expression of p16(INK4α) and Rb proteins in the testosterone-treated Tfm mice. After co-treatment with anastrazole in Tfm mice, these improvement were partly inhibited.
CONCLUSIONPhysiological testosterone replacement can delay cardiomyocyte aging in Tfm mice, an effect that is independent of the AR pathway and in part conversion to estradiol.
Androgen-Insensitivity Syndrome ; metabolism ; Animals ; Cellular Senescence ; Cyclin-Dependent Kinase Inhibitor p16 ; analysis ; Female ; Glutathione Peroxidase ; metabolism ; Male ; Mice ; Myocytes, Cardiac ; physiology ; Receptors, Androgen ; physiology ; Superoxide Dismutase ; metabolism ; Testosterone ; physiology
10.Phosphatidylinositol 3-kinase inhibitor, LY294002, induced senescence-like changes in human diploid fibroblasts.
Shuping LI ; Zongyu ZHANG ; Tanjun TONG
Chinese Medical Journal 2003;116(6):901-905
OBJECTIVETo reveal the role of Phosphatidylinositol 3-kinases (PI3Ks) in regulating human diploid fibroblast (2BS cell) senescence as well as the possible mechanisms involved.
METHODSUsing a PI3Ks specific inhibitor, LY294002, cell cycle, apoptosis, proliferation, senescence association beta-galactosidase staining as well as senescence association CKIs, p16(INK4) and p21(Cip1) protein expressions were all measured in the low passages of 2BS cells.
RESULTSBoth 25 micro mol/L and 50 micro mol/L concentrations of LY294002 could cause a significant decrease in cells entering into S phase, and this cell cycle of G(1) phase arrest was dose-dependent. Meanwhile, LY294002 contributed to apoptosis, caused 2BS cell growth arrest, and activated senescence association beta-galactosidase (P < 0.05). In addition, LY294002 could induce time-course expressions of p16(INK4) and p21(Cip1) in 2BS cell lines.
CONCLUSIONSPI3Ks inhibitor LY294002 could induce senescence-like changes in 2BS cell lines. Two senescence associated CKIs, p16(INK4) and p21(Cip1), might be involved in this senescence phenotype proceeding in 2BS cell lines.
Cells, Cultured ; Cellular Senescence ; drug effects ; Chromones ; pharmacology ; Cyclin-Dependent Kinase Inhibitor p16 ; analysis ; Cyclin-Dependent Kinase Inhibitor p21 ; Cyclins ; analysis ; Dose-Response Relationship, Drug ; Enzyme Inhibitors ; pharmacology ; Fibroblasts ; drug effects ; physiology ; G1 Phase ; drug effects ; Humans ; Morpholines ; pharmacology ; Phosphatidylinositol 3-Kinases ; antagonists & inhibitors