The effects of over-expression of testis-specific expressed gene 1 (TSEG-1) on the viability and apoptosis of cultured spermatogonial GC-1spg cells were investigated, and the immortal spermatogonial cell line GC-1spg (CRL-2053™) was obtained as the cell model in order to explore the function of TSEG-1. We transfected the eukaryotic vector of TSEG-1, named as pEGFP-TSEG-1 into cultured spermatogonial GC-1spg cells. Over-expression of TSEG-1 inhibited the proliferation of GC-1spg cells, and arrested cell cycle slightly at G0/G1 phase. Transfection of TSEG-1 attenuated the transcript levels of Ki-67, PCNA and cyclin D1. In addition, over-expression of TSEG-1 induced early and late apoptosis, and reduced the mitochondrial membrane potential of GC-1spg cells. Moreover, transfection of TSEG-1 significantly enhanced the ratio of Bax/Bcl-2 and transcript levels of caspase 9, and decreased the expression of Fas and caspase 8 in GC-1spg cells. These results indicated over-expression of TSEG-1 suppresses the proliferation and induces the apoptosis of GC-1spg cells, which establishes a basis for further study on the function of TSEG-1.
Animals ; Caspase 8 ; biosynthesis ; genetics ; Cell Line ; Cyclin D1 ; biosynthesis ; genetics ; G1 Phase ; physiology ; Histones ; genetics ; metabolism ; Ki-67 Antigen ; biosynthesis ; genetics ; Male ; Mice ; Proliferating Cell Nuclear Antigen ; biosynthesis ; genetics ; Resting Phase, Cell Cycle ; physiology ; Spermatogonia ; cytology ; metabolism ; bcl-2-Associated X Protein ; biosynthesis ; genetics

Drug resistance is an important character of leukemic stem cells. To explore the mechanism of the chemotherapy resistance of N-cadherin positive leukemia cells, the quiescent state of N-cadherin positive leukemia cells was determined by flow cytometry and the relationship of G(0) phase cell ratio with the chemotherapy resistance was analyzed. After KG1a cells were induced to enter cell cycle, the G(0) phase cell ratio and the sensitivity of cells to VP16 were determined. Finally the quiescent state and drug resistance properties of KG1a cells were determined after inhibiting N-cadherin-mediated cell-cell interaction by EGTA treatment. The results showed that the G(0) phase cell ratio in N-cadherin positive KG1a cells was higher than that in N-cadherin negative KG1a cells. After KG1a cells were induced to enter cell cycle, the G(0) phase cell ratio was decreased significantly and the sensitivity of KG1a cells to VP16 increased. Following EGTA treatment for 24 hours, the G(0) phase cell ratio decreased and the drug-sensitivity was enhanced significantly. It is concluded that N-cadherin-mediated adhesion keeps N-cadherin positive leukemia cells in quiescent state of G(0) phase, thus protect these leukemia cells against VP16 chemotherapy.
Antigens, CD ; metabolism ; Apoptosis ; drug effects ; Cadherins ; metabolism ; Cell Line, Tumor ; Etoposide ; pharmacology ; therapeutic use ; Flow Cytometry ; Humans ; Leukemia, Myeloid, Acute ; drug therapy ; Resting Phase, Cell Cycle ; drug effects

AIMTo investigate the effect of DAPT (gamma-secretase inhibitor) on the growth of human tongue carcinoma cells and to determine the molecular mechanism to enable the potential application of DAPT to the treatment of tongue carcinoma.

METHODOLOGYHuman tongue carcinoma Tca8113 cells were cultured with DAPT. Cell growth was determined using Indigotic Reduction method. The cell cycle and apoptosis were analyzed by flow cytometry. Real-time PCR and Immuno-Fluorescence (IF) were employed to determine the intracellular expression levels.

RESULTSDAPT inhibited the growth of human tongue carcinoma Tca8113 cells by inducing G0-G1 cell cycle arrest and apoptosis. The mRNA levels of Hairy/Enhancer of Split-1 (Hes-1), a target of Notch activation, were reduced by DAPT in a dose-dependent manner. Coincident with this observation, DAPT induced a dose-dependent promotion of constitutive Caspase-3 in Tca8113 cells.

CONCLUSIONDAPT may have a therapeutic value for human tongue carcinoma. Moreover, the effects of DAPT in tumor inhibition may arise partly via the modulation of Notch-1 and Caspase-3.

Amyloid Precursor Protein Secretases ; antagonists & inhibitors ; Antineoplastic Agents ; administration & dosage ; pharmacology ; Apoptosis ; drug effects ; Basic Helix-Loop-Helix Transcription Factors ; drug effects ; Carcinoma ; pathology ; Caspase 3 ; drug effects ; Cell Line, Tumor ; Cell Membrane ; drug effects ; Cell Nucleus ; drug effects ; Cyclin D1 ; drug effects ; Dipeptides ; administration & dosage ; pharmacology ; Dose-Response Relationship, Drug ; G1 Phase ; drug effects ; Homeodomain Proteins ; drug effects ; Humans ; Receptor, Notch1 ; drug effects ; Repressor Proteins ; drug effects ; Resting Phase, Cell Cycle ; drug effects ; Tongue Neoplasms ; pathology ; Transcription Factor HES-1

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

OBJECTIVE: To explore the effect of LRRC4, a glioma suppressive gene, on blocking U251 cells in G0/G1 by MAPK signaling pathway. METHODS: LRRC4 was transfected into U251 cells, and at 24 hour of post-transfection, cells were split at a 1:3 dilution, challenged with 500 microg /mL G418 and formed a stable transfected clone pool. RT-PCR, Northern blot and Western blot were used to identify the stable transfectants. ERK, JNK and P38 expression changes were analyzed by Western blot. FACS analysis, Luciferase reporter gene assay and Western blot were used to detect the cell cycle and cyclin D1. RESULTS: LRRC4 down-regulated the expression of phosphorylated ERK2 and up-regulated the expression of total protein JNK2 (a key molecule of MAPK signaling pathway) and phosphorylated c-Jun. LRRC4 decreased the expression of mutation P53, cyclin D1 activation and its expression. U251 cells were blocked in G0/G1 by LRRC4. CONCLUSION LRRC4 can decrease JNK2, up-regulate the phosphoralated c-Jun, down-regulate mutant P53 and cyclin D1, and therefore block U251 cells in G0/G1.
Blotting, Northern ; Blotting, Western ; Cell Line, Tumor ; Cyclin D1 ; metabolism ; Flow Cytometry ; G1 Phase ; genetics ; physiology ; Glioma ; genetics ; metabolism ; pathology ; Humans ; Luciferases ; genetics ; metabolism ; MAP Kinase Signaling System ; genetics ; physiology ; Mitogen-Activated Protein Kinase Kinases ; metabolism ; Nerve Tissue Proteins ; genetics ; metabolism ; physiology ; RNA, Messenger ; biosynthesis ; genetics ; Recombinant Fusion Proteins ; genetics ; metabolism ; Resting Phase, Cell Cycle ; genetics ; physiology ; Reverse Transcriptase Polymerase Chain Reaction ; Transfection

AIMTo evaluate the antiproliferative activity of contragestazol (DL111-IT) on the human prostate cancer cell line PC3 in vitro and in vivo and to elucidate its potential molecular mechanisms.

METHODSThe cell killing ability of DL111-IT was measured by the 3-(4,5-dimethylthia-zol,2-yl)-2,5-diphenyltetrazolium bromide (MTT) reagent assay method and the tumor xenograft model. The cell cycle was analyzed by flow cytometry and protein expression, including retinoblastoma (pRb), cyclin-dependent kinase 4 (CDK4) and cyclin D1, was detected by Western blotting.

RESULTSDL111-IT exhibited high efficiency on cell growth inhibition of the human androgen-independent prostate cancer cell line PC3. The drug concentration that yielded 50% cell inhibition (IC50 value) was 9.9 mg/mL. In the PC3 tumor xenograft study, DL111-IT (1.25 mg/kg-20.0 mg/kg) given once a day for 10 days significantly inhibited tumor growth, with the inhibition rate ranging from 21% to 50%. Flow cytometric analysis indicated that DL111-IT could cause G1 arrest in the PC3 cell line, but not apoptosis. DL111-IT enhanced pRb expression and down-regulated CDK4 and cyclin D1 expression, suggesting that cell cycle regulation might contribute to the anticancer property of DL111-IT.

CONCLUSIONDL111-IT inhibits the proliferation of human androgen-independent prostate cancer cell line PC3 in vitro and in vivo by a cell cycle regulation pathway.

Androgens ; pharmacology ; Animals ; Cell Division ; drug effects ; Cell Line, Tumor ; Cyclin D1 ; metabolism ; Cyclin-Dependent Kinase 4 ; metabolism ; Dose-Response Relationship, Drug ; Female ; G1 Phase ; drug effects ; Humans ; Immunosuppressive Agents ; pharmacology ; In Vitro Techniques ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Neoplasm Transplantation ; Prostatic Neoplasms ; drug therapy ; pathology ; Resting Phase, Cell Cycle ; drug effects ; Retinoblastoma Protein ; metabolism ; Transplantation, Heterologous ; Triazoles ; pharmacology

Animals ; Cells, Cultured ; DNA ; analysis ; Flow Cytometry ; methods ; Fluorescence ; G1 Phase ; Resting Phase, Cell Cycle

AIMTo study the effects of 9-cis-retinoic acid (9-cis-RA) on cell cycle and expression of cyclin D1 and cdk4 in lung cancer cells.

METHODS9-cis-RA (1 x 10(-6) mol.L-1) was used to treat lung cancer cells for 24 h; Flow cytometry (FCM) was used to detect the percent of G0/G1 phase and S phase cells of three groups including blank control, DMSO control and 9-cis-RA groups; RT-PCR was used to analyze the expression changes of cyclin D1 and cdk4 before and after treatment with 9-cis-RA in lung cancer cells.

RESULTSThe percent of G0/G1 phase cells of 9-cis-RA groups was significantly higher than that of the control groups (P < 0.01 or P < 0.05) and the percent of S phase cells of 9-cis-RA groups was lower than that of the control groups (P < 0.01 or P < 0.05); the expression of cyclin D1 of PG, SPC-A1 and L78 cells was decreased (P < 0.01) and the expression of cdk4 of PG, A549 and L78 cells was also decreased (P < 0.01) after treatment with 9-cis-RA.

CONCLUSIONMost of the proliferation and the expression of cyclin D1 and cdk4 of PG, A549, SPC-A1 and L78 were inhibited by 9-cis-RA.

Adenocarcinoma ; metabolism ; pathology ; Antineoplastic Agents ; pharmacology ; Carcinoma, Squamous Cell ; metabolism ; pathology ; Cell Division ; drug effects ; Cell Line, Tumor ; Cyclin D1 ; biosynthesis ; Cyclin-Dependent Kinase 4 ; Cyclin-Dependent Kinases ; biosynthesis ; G1 Phase ; drug effects ; Humans ; Lung Neoplasms ; metabolism ; pathology ; Proto-Oncogene Proteins ; Resting Phase, Cell Cycle ; drug effects ; S Phase ; drug effects ; Tretinoin ; pharmacology

OBJECTIVETo study the inhibitory effects of ciglitazone, a synthetic ligand of peroxisome proliferator-activated receptors (PPAR), on human lung cancer growth in vitro and in vivo and its mechanisms.

METHODSHuman lung cancer A549 cells cultured in vitro were treated with different concentrations of ciglitazone. The proliferative activity and cell cycle of A549 cells were determined by MTT assay and flow cytometry. Expression of PPARgamma protein was detected by Western blot. A549 cells (1 x 10(6) cells/nude mouse) were inoculated subcutaneously into nude mice, which were randomly divided into two groups, 10 in each: control group (group A) and ciglitazone treated group (group B). When the tumors grew to a size with diameter around 1 cm, ciglitazone 100 microl (100 micromol/L) was intratumorally injected every other day in group B mice. A total of 15 injections were given. Mice in group A were similarly treated with normal saline. One month later, tumors were excised and weighed. Expression of cyclin D1 and p21 protein were detected by immunohistochemistry and Western blot.

RESULTSGrowth of A549 cells was significantly inhibited in group B in a dose-dependent and time-dependent fashion as compared with that in group A. Most of the ciglitazone-treated cells arrested in G(1)/G(0) phase and the expression of PPARgamma protein was markedly up-regulated. The tumor weights in group A was (2.79 +/- 0.33) g and that in group B was (1.51 +/- 0.40) g, with an inhibition rate of 47.0%. The expression level of cyclin D1 in group A was significantly higher than that in group B, while the expression level of p21 protein in group A was significantly lower than that in group B.

CONCLUSIONCiglitazone can effectively inhibit the growth of human lung cancer A549 and induce its differentiation by cell cycle arrest via PPARgamma activation.

Animals ; Cell Differentiation ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cyclin D1 ; metabolism ; Cyclin-Dependent Kinase Inhibitor p21 ; metabolism ; Dose-Response Relationship, Drug ; Female ; G1 Phase ; Gene Expression Regulation, Neoplastic ; Humans ; Lung Neoplasms ; drug therapy ; metabolism ; pathology ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; PPAR gamma ; metabolism ; Random Allocation ; Resting Phase, Cell Cycle ; Thiazolidinediones ; pharmacology ; therapeutic use ; Time Factors

OBJECTIVETo explore the influence of different nutritional support routes on the intestinal mucosal epithelial cell cycle in burned rats.

METHODSSixty-six Wistar rats inflicted with 30% TBSA III degree burns on the back were employed as the model and were randomly divided into enteral feeding group (EF) and intravenously parenteral nutrition group (PN). Equal volume of nutritional support fluid containing predetermined equal amount of calories and nitrogen was applied via feeding or intravenously infusion through external jugular vein. The indices were observed on 6, 12, 24, 48 and 72 postburn hours (PBHs) with the reference to those in 6 normal rats. The intestinal epithelial cell cycle in jejunal and ileal mucous membrane was analyzed by flow cytometry. Western blotting method was employed in the examination of the expression of cyclin D1, E and that of cyclin dependent kinase (CDK)2 and CDK4.

RESULTS(1) lntestinal mucosal epithelial G0/G1 ratio in jejunum in EF group was significantly lower than that in PN group at 72 PBHs (P < 0.05). While the ratio in ileum in EF was obviously higher than that in PN groups at 6, 12, 48 and 72 PBHs (P < 0.05). (2) The cell percentage of S phase in EF group was evidently higher than that in PN group (P < 0.05 - 0.01) at 48 and 72 PBHs. (3) Intestinal mucosal cyclin D1 expression increased significantly in EF group at 24 PBHs and in PN group at 48 PBHs (P < 0.05) and which in EF group was obviously higher than that in PN group at 72 PBHs (P < 0.05). (4) The expression of the intestinal mucosal cyclin E in EF group at 72 PBHs was evidently higher than the control value and that in PN group (P < 0.05). (5) The expression of CDK2 exhibited no obvious difference among PN,EF and control group (P < 0.05). The CDK4 expression in EF group increased obviously at 72 PBHs (P < 0.05).

CONCLUSIONEarly postburn enteral feeding was beneficial to the progression of intestinal mucosal epithelial cell cycle and to the repairing and renovation of injured intestinal mucosal membrane. Cyclin and CDK might be important in the modulation of the intestinal mucosal epithelial cell cycle.

Animals ; Burns ; metabolism ; pathology ; CDC2-CDC28 Kinases ; Cell Cycle ; physiology ; Cyclin D1 ; metabolism ; Cyclin-Dependent Kinase 2 ; Cyclin-Dependent Kinase 4 ; Cyclin-Dependent Kinases ; metabolism ; Disease Models, Animal ; Enteral Nutrition ; Female ; G1 Phase ; physiology ; Intestinal Mucosa ; metabolism ; pathology ; Male ; Protein-Serine-Threonine Kinases ; metabolism ; Proto-Oncogene Proteins ; Rats ; Rats, Wistar ; Resting Phase, Cell Cycle ; physiology ; S Phase ; physiology