OBJECTIVECytotoxin-associated protein (CagA) of H. pylori has been confirmed to be closely associated with gastric inflammation and tumorigenesis, but the mechanism behind it is little understood. In this study, we try to determine roles of CagA(+) strain in activating PI3K/Akt1 signaling pathway, and affecting expression of p21(WAF1/CIP1) and p27(KIP1), and also in releasing IL-8 in host cells.

METHODSAkt1 phosphorylation and IL-8 levels of CagA(+) and CagA⁻ strain infected AGS cells were detected by ELISAs. Two quantitative RT-PCRs were established to measure p21(WAF1/CIP1) and p27(KIP1) mRNA levels in the CagA(+) and CagA⁻ strain infected cells. LY294002, an inhibitor of PI3K/Akt pathway, was used to define effect of the pathway in IL-8 release.

RESULTSCagA(+) strain could induce an obvious elevation of Akt1 phosphorylation in the infected AGS cells while CagA? strain failed to do so. The CagA(+) H. pylori strain infected AGS cells showed significant drops both in p21(WAF1/CIP1) and p27(KIP1) mRNA levels, whereas the CagA⁻ H. pylori strain caused a remarkable increase in p21(WAF1/CIP1) mRNA without affecting p27(KIP1) gene transcription in the AGS cells. Both the CagA(+) and CagA⁻ H. pylori strains enabled AGS cells to produce close elevated levels of IL-8, and the LY294002 block resulted in unexpected elevations of IL-8 levels.

CONCLUSIONSCagA can activate PI3K/Akt1 pathway that plays an inhibitory role in IL-8 release in H. pylori infected AGS cells. Activation of PI3K/Akt1 pathway and subsequent negative regulation of p21(WAF1/CIP1) and p27(KIP1) expression might be involved in CagA-associated carcinogenesis.

Antigens, Bacterial ; genetics ; physiology ; Bacterial Proteins ; genetics ; physiology ; Cell Line ; Cyclin-Dependent Kinase Inhibitor p21 ; biosynthesis ; Cyclin-Dependent Kinase Inhibitor p27 ; Gastric Mucosa ; cytology ; enzymology ; microbiology ; Helicobacter pylori ; metabolism ; pathogenicity ; physiology ; Humans ; Interleukin-8 ; secretion ; Intracellular Signaling Peptides and Proteins ; metabolism ; Phosphatidylinositol 3-Kinases ; metabolism ; Phosphorylation ; Proto-Oncogene Proteins c-akt ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Signal Transduction ; Transcription, Genetic ; Virulence

Alternative Splicing ; Apoptosis ; Cell Cycle ; Cell Proliferation ; Cyclin-Dependent Kinase Inhibitor p21 ; metabolism ; Hep G2 Cells ; Humans ; Inhibitor of Growth Protein 1 ; Intracellular Signaling Peptides and Proteins ; genetics ; metabolism ; physiology ; Nuclear Proteins ; genetics ; metabolism ; physiology ; Plasmids ; Protein Isoforms ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Recombinant Proteins ; genetics ; metabolism ; Transfection ; Tumor Suppressor Protein p53 ; metabolism ; Tumor Suppressor Proteins ; genetics ; metabolism ; physiology ; bcl-2-Associated X Protein ; metabolism

OBJECTIVETo investigate the mechanism by which recombinant adenovirus (Ad)-mediated mutations of hypoxia inducible factor 1alpha (Ad-HIF-1alpha-Ala564-Ala803) regulates cell apoptosis.

METHODSLoVo cells were infected with recombinant Ad-HIF-1alpha-Ala564-Ala803 and control virus Ad-lacZ under normoxia condition. Real-time PCR was used to detect HIF-1alpha and p21WAF1/CIP1 mRNA expressions at different time points. Western blotting was employed to verify HIF-1alpha and p21WAF1/CIP1 protein expression. Hoechst 33342 flourescein staining was performed to observe the ratio of apoptotic LoVo cells.

RESULTSThe expression levels of HIF-1alpha mRNA and protein increased after infection with Ad-HIF-1alpha- Ala564-Ala803, accompanied by an increase in p21WAF1/CIP1 mRNA and protein expressions. The apoptotic ratio was significantly higher in LoVo cells infected with recombinant Ad-HIF-1alpha-Ala564-Ala803 (16.2%) than in the control cells (5.5%, P=0.00).

CONCLUSIONHIF-1alpha may induce cell cycle arrest by up-regulating p21WAF1/CIP1 expressions to promote LoVo cell apoptosis.

Adenoviridae ; genetics ; Apoptosis ; genetics ; physiology ; Blotting, Western ; Cell Line, Tumor ; Colonic Neoplasms ; genetics ; metabolism ; pathology ; Cyclin-Dependent Kinase Inhibitor p21 ; biosynthesis ; genetics ; Genetic Vectors ; genetics ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit ; biosynthesis ; genetics ; Mutagenesis, Site-Directed ; Mutation ; RNA, Messenger ; biosynthesis ; genetics ; Recombinant Proteins ; biosynthesis ; Reverse Transcriptase Polymerase Chain Reaction ; Transfection

OBJECTIVETo observe the inhibitory effect of cyclin-dependent kinase inhibitor p21 on regulation of survivin transcription in human liver cancer HepG cells, and explore the related mechanisms.

METHODSDoxorubicin (DOX) was used to treat HepG cells. Eukaryotic vector pEGFP-C2-p21 was transfected into HepG cells by lipofectamine and positive clones were screened out by G418. The mRNA expression of p21, p53 and survivin were detected by real-time fluorescent quantitative polymerase chain reaction (RQ-PCR). Flow cytometry was used to determine the cell cycle phases, and reverse transcription polymerase chain reaction (RT-PCR) was used to measure the levels of E2F-1 or p300.

RESULTSAfter treatment with DOX, the expression of p53 and p21 was increased, whereas that of survivin was reduced during 24 hours of the treatment. After transfection the p21 level was 2100.1-fold or 980.9-fold enhanced in comparison with that in HepG2 cells or HepG2-pEGFP cells. Survivin level was markedly down-regulated to 0.5% or 0.6% relative to that in the other two groups, nevertheless, significant p53 changes were not observed. Overexpression of p21 resulted in G1/G0 phase arrest (F = 31.59, P < 0.01), meanwhile, E2F-1 mRNA or p300 mRNA were less expressed compared with that in the other controls (F(E2F-1) = 125.28, P < 0.05; Fp300 = 46.01, P < 0.01).

CONCLUSIONp21 could be a potential mediator of survivin suppression at transcription level in HepG2 cells, which might be through the block at G1/G0 phase and down-regulation of transcription factors E2F-1 and p300.

Antibiotics, Antineoplastic ; pharmacology ; Cyclin-Dependent Kinase Inhibitor p21 ; genetics ; metabolism ; physiology ; Doxorubicin ; pharmacology ; E2F1 Transcription Factor ; genetics ; metabolism ; G1 Phase ; Gene Expression Regulation, Neoplastic ; Hep G2 Cells ; Humans ; Inhibitor of Apoptosis Proteins ; Microtubule-Associated Proteins ; genetics ; metabolism ; RNA, Messenger ; metabolism ; Resting Phase, Cell Cycle ; Transfection ; Tumor Suppressor Protein p53 ; genetics ; metabolism ; p300-CBP Transcription Factors ; genetics ; metabolism

OBJECTIVETo observe the effect of Bushen Yiqi Hexue recipe on endometrial P21 and PCNA expression of embryo implantation dysfunctional mice.

METHODThe expression of P21 protein was analyzed by means of immunoblot and immunohistochemical method. The expression of PCNA protein was analyzed by means of immunohistochemical method.

RESULTThe expression and disappearance of P21 and PCNA protein in endometrium of embryo implantation dysfunctional mice lagged behind in a temporal and cell specific manner. Bushenyiqihexue recipe could improve the expression of P21 and PCNA protein in a temporal and cell specific manner, and then improve the decidual reaction of endometrium of embryo implantation dysfunctional mice.

CONCLUSIONImproving the expression of P21 and PCNA protein in endometrium and eventually regulating endometrium decidual reaction is one of the mechanisms of improved embryo implantaion by Bushen Yiqi Hexue recipe.

Animals ; Cyclin-Dependent Kinase Inhibitor p21 ; metabolism ; Drug Combinations ; Drugs, Chinese Herbal ; isolation & purification ; pharmacology ; Embryo Implantation ; drug effects ; physiology ; Endometrium ; metabolism ; Female ; Immunoblotting ; Immunohistochemistry ; Mice ; Plants, Medicinal ; chemistry ; Proliferating Cell Nuclear Antigen ; metabolism

In vascular smooth muscle cells (VSMCs), induction of the heme oxygenase-1 (HO-1) confers vascular protection against cellular proliferation mainly via its up-regulation of the cyclin-dependent kinase inhibitor p21(WAF1/CIP1) that is involved in negative regulation of cellular proliferation. In the present study, we investigated whether the phytochemical curcumin and its metabolite tetrahydrocurcumin could induce HO-1 expression and growth inhibition in rat VSMCs and, if so, whether their antiproliferative effect could be mediated via HO-1 expression. At non-toxic concentrations, curcumin possessing two Michael-reaction acceptors induced HO-1 expression by activating antioxidant response element (ARE) through translocation of the nuclear transcription factor E2-related factor-2 (Nrf2) into the nucleus and also inhibited VSMC growth triggered by 5% FBS in a dose-dependent manner. In contrast, tetrahydrocurcumin lacking Michael-reaction acceptor showed no effect on HO-1 expression, ARE activation and VSMC growth inhibition. The antiproliferative effect of curcumin in VSMCs was accompanied by the increased expression of p21(WAF1/CIP1). Inhibition of VSMC growth and expression of p21(WAF1/CIP1) by curcumin were partially, but not completely, abolished when the cells were co- incubated with the HO inhibitor tin protoporphyrin. In human aortic smooth muscle cells (HASMCs), curcumin also inhibited growth triggered by TNF-alpha and increased p21(WAF1/CIP1) expression via HO-1-dependent manner. Our findings suggest that curcumin has an ability to induce HO-1 expression, presumably through Nrf2-dependent ARE activation, in rat VSMCs and HASMCs, and provide evidence that the antiproliferative effect of curcumin is considerably linked to its ability to induce HO-1 expression.
Active Transport, Cell Nucleus ; Animals ; Aorta/cytology ; Cell Nucleus/metabolism ; Cell Proliferation/*drug effects ; Cells, Cultured ; Curcumin/analogs & derivatives/*pharmacology ; Cyclin-Dependent Kinase Inhibitor p21/biosynthesis/metabolism ; Gene Expression Regulation ; Heme Oxygenase (Decyclizing)/biosynthesis/genetics/*physiology ; Heme Oxygenase-1/biosynthesis/genetics/*physiology ; Humans ; Metalloporphyrins/pharmacology ; Muscle, Smooth, Vascular/drug effects/*physiology ; Myocytes, Smooth Muscle/drug effects/*physiology ; NF-E2-Related Factor 2/metabolism ; Protoporphyrins/pharmacology ; Rats ; Regulatory Sequences, Nucleic Acid ; Response Elements ; Tumor Necrosis Factor-alpha/pharmacology

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

OBJECTIVETo investigate the biological function of RIG-G gene by establishing a cell line stably expressing RIG-G protein.

METHODSEctopic RIG-G gene was transfected into U937 cells by using Tet-off expression system. Changes before and after RIG-G expression were detected for cell growth, cell morphology, cell surface antigen and cell cycle regulating proteins.

RESULTSRIG-G protein arrested the cells at G0/G1 phase and inhibited cell growth by increasing the cell cycle inhibitors P21 and P27. As compared to that in control group, the proportion of cells at G0/G1 phase in RIG-G-expressing cell group increased from (43.9 +/- 5.6)% to (63.9 +/- 2.3)% (P < 0.01). The rate of growth inhibition was (68.7 +/- 0.2)%. In addition, an increase in CD11C expression [(61.3 +/- 1.1)% vs. (18.0 +/- 0.4)% (P < 0.01)] and in cells with morphologic features of partial differentiation (smaller cell size, reduced nucleus-cytoplasm ratio, notched nucleus and coarse chromatin) was also observed in RIG-G-expressing cells.

CONCLUSIONSRIG-G has potential abilities to inhibit cell proliferation and promote cell differentiation.

Cell Cycle ; genetics ; Cell Differentiation ; Cell Proliferation ; Cyclin-Dependent Kinase Inhibitor p21 ; genetics ; metabolism ; Cyclin-Dependent Kinase Inhibitor p27 ; genetics ; metabolism ; Humans ; Intracellular Signaling Peptides and Proteins ; genetics ; physiology ; Plasmids ; genetics ; Transfection ; U937 Cells

OBJECTIVETo evaluate the relationship between mammalian target of rapamycin (mTOR) signaling pathway and histone acetylation in cell survival, cell cycle, gene expression and protein level on human gastric cancer cells.

METHODSHuman gastric cancer cell lines, MKN45 and SGC7901 were treated with trichostatin A, rapamycin and/or LY294002, a PI3K inhibitor. Cell viability was analyzed by methylthiazolyl tetrazolium. Cell cycle distribution was evaluated by flow cytometry. The transcription level of p21(WAF1) gene was detected by using real-time polymerase chain reaction. Proteins were detected by Western blotting.

RESULTSCell viability remarkably reduced after treatment by more than two drugs (P< 0.01). Through flow cytometry assessment, MKN45 cells were arrested in G2 phase (P< 0.05), while SGC7901 cells were in G2 or G1 phase (P< 0.05) whether treated with single or more than two drugs. The expression of p21(WAF1) mRNA was remarkably increased in the gastric cancer cells treated with conjoined drugs (P< 0.01). Phosphorylation of Akt, p70S6K and 4E-BP1 was significantly reduced in cells treated with conjoined drugs (P< 0.01). And histone acetylation of H4/H3 was also increased in cells treated with conjoined drugs (P< 0.01).

CONCLUSIONmTOR singnaling pathway has an important relationship with histone acetylation in gastric cancer cell lines. There is a co-effect of mTOR inhibitor and histone deacetylase inhibitor on gastric cancer cells.

Acetylation ; drug effects ; Adaptor Proteins, Signal Transducing ; metabolism ; Blotting, Western ; Cell Cycle ; drug effects ; Cell Line, Tumor ; Cell Survival ; drug effects ; Chromones ; pharmacology ; Cyclin-Dependent Kinase Inhibitor p21 ; genetics ; Flow Cytometry ; Histones ; metabolism ; Humans ; Hydroxamic Acids ; pharmacology ; Morpholines ; pharmacology ; Phosphoproteins ; metabolism ; Phosphorylation ; drug effects ; Polymerase Chain Reaction ; Protein Kinases ; metabolism ; Proto-Oncogene Proteins c-akt ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Ribosomal Protein S6 Kinases, 70-kDa ; metabolism ; Signal Transduction ; drug effects ; physiology ; Sirolimus ; pharmacology ; Stomach Neoplasms ; metabolism ; pathology ; physiopathology ; TOR Serine-Threonine Kinases

SC-560, a strucutral analogue of celecoxib, induces growth inhibition in a wide range of human cancer cells in a cyclooxygenase (COX)-independent manner. Since SC-560 suppresses the growth of cancer cells mainly by inducing cell cycle arrest, we sought to examine the role of p21CIP1, a cell cycle regulator protein, in the cellular response against SC-560 by using p21(+/+)and p21(-/-)isogenic HCT116 colon carcinoma cells. In HCT116 (p21(+/+)) cells, SC-560 dose-dependently induced growth inhibition and cell cycle arrest at the G1 phase without significant apoptosis induction. SC-560-induced cell cycle arrest was accompanied by upregulation of p21CIP1. However, the extent of SC-560-induced accumulation at the G1 phase was approximately equal in the p21(+/+)and the p21(-/-)cells. Nonetheless, the growth inhibition by SC-560 was increased in p21(-/-)cells than p21(+/+)cells. SC-560-induced reactive oxygen species (ROS) generation did not differ between p21(+/+)and p21(-/-)cells but the subsequent activaton of apoptotic caspase cascade was more pronounced in p21(-/-)cells compared with p21(+/+)cells. These results suggest that p21CIP1 blocks the SC-560-induced apoptotic response of HCT116 cells. SC-560 combined with other therapy that can block p21 CIP1 expression or function may contribute to the effective treatment of colon cancer.
Reactive Oxygen Species/metabolism ; Pyrazoles/*pharmacology ; Mutation ; Immunoblotting ; Humans ; HCT116 Cells ; Genotype ; Flow Cytometry ; Dose-Response Relationship, Drug ; Cyclooxygenase Inhibitors/pharmacology ; Cyclin-Dependent Kinase Inhibitor p21/genetics/metabolism/*physiology ; Colonic Neoplasms/genetics/metabolism/pathology ; Cell Survival/drug effects ; Cell Proliferation/drug effects ; Cell Differentiation/drug effects ; Cell Cycle Proteins/metabolism ; Cell Cycle/drug effects ; Apoptosis/drug effects ; Antineoplastic Agents/pharmacology