The study was designed to examine the effect of glucose on the expression of c-myc gene in cultured RINm5F cells. After monolayer culture was established in RPMI 1640 media supplemented with 10% fetal calf serum (FCS), the cells were cultured in various concentrations of glucose and 1 or 10% FCS for another 24 hours. A mRNA was extracted from the cultured cells by a single step method, and Northern analysis was done to detect RNA band. A 0.5 kilobase single band was detected as c-myc mRNA. The expression of c-myc gene mRNA was reduced with increased concentration of glucose with 1% FCS. However, supplementation of 10% FCS abolished the effect of glucose on expression of c-myc gene. These findings suggested that glucose in conjunction with other growth promoting factors played an important role in expression of oncogene and cell growth in RINm5F cells.
Animals ; Cell Line ; Gene Expression Regulation, Neoplastic/drug effects ; Genes, myc/*drug effects ; Glucose/*pharmacology ; Insulinoma/*genetics ; Pancreatic Neoplasms/*genetics ; Rats ; Tumor Cells, Cultured

This study was aimed to observe the effects of emodin on apoptosis and cell cycle related genes in human myeloid leukemia cell line U937 cells. U937 cells were exposed to 60 µmol/L emodin for 24, 48, 72 h. The expressions of C-MYC, h-TERT, PIM-2, Survivin, wild type P53, P21, TGF β-1 and MCL-1 genes before and after treatment with emodin were determined and quantitated by using reverse transcriptase-polymerase chain reaction (RT-PCR). The results showed that the expressions of C-MYC, h-TERT, PIM-2, Survivin in treated U937 cells decreased, but the expressions of WTp53, P21 and TGFβ1 increased, while the expression of MCL-1 gene had no obvious change. It is concluded that multiple pathways may be involved in the processes of emodin-induced U937 cell apoptosis.
Apoptosis ; drug effects ; Apoptosis Regulatory Proteins ; Cell Cycle ; drug effects ; Cell Proliferation ; Emodin ; pharmacology ; Genes, myc ; Humans ; Reverse Transcriptase Polymerase Chain Reaction ; U937 Cells

OBJECTIVETo study the effect of astragalus injection on U937 leukemia cells proliferation and apoptosis and relevant molecular mechanisms.

METHODSLeukemia cell line U937 cells were treated with different concentrations of astragalus (62.5, 125, 250, 500, 1 000 μg/mL). The U937 cells without astragalus treatment were used as the control group. The ability of cell proliferation was measured by MTT method. Flow cytometry was used to explore cell apoptosis. The cell morphology changes were observed under a fluorescent microscope by dyeing Hoechst33258. mRNA expression of c-myc and p27 in U937 cells which was exposed in 1 000 μg/mL astragalus after 0, 12, 24 and 48 hours was detected by reverse transcription polymerase chain reaction (RT-PCR).

RESULTSVarious concentrations of astragalus injection inhibited U937 cell proliferation effectively compared with the control group (P<0.05). They also induced U937 cells apoptosis and the apoptosis rate reached to (63 ± 4)% in the 1 000 μg/mL astragalus treatment group. mRNA expression level of c-myc was gradually declined and p27 mRNA expression was gradually increased with astragalus treatment time (P<0.01).

CONCLUSIONSAstragalus injection may inhibit proliferation and induce apoptosis of leukemia cell line U937 in vitro. This contributes to down-regulation of c-myc expression and up-regulation of p27 expression.

Apoptosis ; drug effects ; Astragalus Plant ; Cell Proliferation ; drug effects ; Cyclin-Dependent Kinase Inhibitor p27 ; genetics ; Genes, myc ; Humans ; Injections ; U937 Cells

OBJECTIVETo construct the recombinant adenovirus encoding antisense c-myc fragment and to investigate its effect on the chemotherapy sensitivity of osteosarcoma MG-63 cells to cisplatin.

METHODSThe recombinant adenovirus (Ad-Asc-myc) encoding antisense c-myc fragment was constructed by cloning c-myc cDNA of about 720 base pairs in a reverse direction into adenovirus vector, then undergoing recombination, amplifying and being complemented in vivo. The osteosarcoma MG-63 cells were transfected by the Ad-Asc-myc in vitro, and Wright staining, Acridine Orange staining, Western Blot, MTT, Flow Cytometry (FCM) were used to study cell morphology, expression of c-myc protein, tumor cell proliferation in vitro, apoptosis and cell cycle change.

RESULTSAd-Asc-myc encoding antisense c-myc fragment was obtained with the titer of 2 x 10(9) pfu/ml. Ad-Asc-myc down-regulated the expression of c-myc protein after transfected MG-63 cells for 48 h, combined with the treatment of 2.0, 5.0 microg/ml cisplatin for 2 h could inhibit tumor cells proliferation in vitro by 33.4% and 54.2% respectively, which were significantly difference compared with control recombinant adenovirus (Ad-LacZ) groups (P < 0.05). Acridine Orange staining and FCM analysis showed that Ad-Asc-myc could induce apoptosis of transfected cells, which was enhanced by the treatment of cisplatin cell. Cycle analysis showed that obvious G2/M phase arrested in transfected cells.

CONCLUSIONAd-Asc-myc increases the chemotherapy sensitivity of osteosarcoma MG-63 cells to cisplatin as well as induced apoptosis.

Adenoviruses, Human ; genetics ; Antineoplastic Agents ; pharmacology ; Apoptosis ; drug effects ; genetics ; Cisplatin ; pharmacology ; DNA, Antisense ; genetics ; Genes, myc ; Genetic Vectors ; Humans ; Osteosarcoma ; genetics ; metabolism ; pathology ; Proto-Oncogene Proteins c-myc ; metabolism ; Recombination, Genetic ; Transfection ; Tumor Cells, Cultured

OBJECTIVETo study the effects of selenium on DNA damage, apoptosis and c-myc, c-fos, and c-jun expression in rat hepatocytes.

METHODSSodium selenite at the doses of 5, 10, and 20 micromol/kg was given to rats by i.p. and there were 5 male SD rats in each group. Hepatocellular DNA damage was detected by single cell gel electrophoresis (or comet assay). Hepatocellular apoptosis was determined by TUNEL (TdT-mediated dUTP nick end labelling) and flow cytometry. C-myc, c-fos, and c-jun expression in rat hepatocytes were assayed by Northern dot hybridization. C-myc, c-fos, and c-jun protein were detected by immunohistochemical method.

RESULTSAt the doses of 5, 10, and 20 micromol/kg, DNA damage was induced by sodium selenite in rat hepatocytes and the rates of comet cells were 34.40%, 74.80%, and 91.40% respectively. Results also showed an obvious dose-response relationship between the rates of comet cells and the doses of sodium selenite (r=0.9501, P<0.01). Sodium selenite at the doses of 5, 10, and 20 micromol/kg caused c-myc, c-fos, and c-jun overexpression obviously. The positive brown-yellow signal for proteins of c-myc, c-fos, and c-jun was mainly located in the cytoplasm of hepatocytes with immunohistochemical method. TUNEL-positive cells were detected in selenium-treated rat livers. Apoptotic rates (%) of selenium-treated liver cells at the doses of 5, 10, and 20 micromol/kg were (3.72 +/- 1.76), (5.82 +/- 1.42), and (11.76 +/- 1.87) respectively, being much higher than those in the control. Besides an obvious dose-response relationship between apoptotic rates and the doses of sodium selenite (r=0.9897, P<0.01), these results displayed a close relationship between DNA damage rates and apoptotic rates, and the relative coefficient was 0.9021, P<0.01.

CONCLUSIONSelenium at 5-20 micromol/kg can induce DNA damage, apoptosis, and overexpression of c-myc, c-fos, and c-jun in rat hepatocytes.

Animals ; Apoptosis ; drug effects ; Blotting, Northern ; Comet Assay ; DNA Damage ; Dose-Response Relationship, Drug ; Genes, fos ; drug effects ; genetics ; Genes, jun ; drug effects ; genetics ; Genes, myc ; drug effects ; genetics ; Hepatocytes ; drug effects ; pathology ; Male ; Nucleic Acid Hybridization ; Rats ; Rats, Sprague-Dawley ; Selenium ; pharmacology ; Sodium Selenite ; pharmacology

OBJECTIVETo investigate the molecular biological mechanism of endothelial cell proliferation induced by bFGF.

METHODSCultured rat myocardial microvascular endothelial cells were stimulated with bFGF of various concentrations. By northern blot analysis, the levels of c-myc mRNA expression were detected.

RESULTSThe expression of c-myc mRNA in the bFGF-treated groups increased (P < 0.05) with a dose- and stimulating time-dependent manner. The c-myc mRNA expression reached to a peak level at 2 hours.

CONCLUSIONSC-myc expression may be an important component in controlling the transit of cells through the cell cycle.

Animals ; Cell Division ; drug effects ; Cells, Cultured ; Dose-Response Relationship, Drug ; Endothelium, Vascular ; cytology ; drug effects ; metabolism ; Fibroblast Growth Factor 2 ; pharmacology ; Gene Expression Regulation ; drug effects ; Genes, myc ; genetics ; RNA, Messenger ; genetics ; metabolism ; Rats ; Rats, Sprague-Dawley ; Time Factors

BACKGROUNDEpigallocatechin-3-gallate (EGCG) has been demonstrated to have anti-neoplastic activity, but the effective concentration of EGCG and its possible mechanisms are uncertain. The study on the killing effects of EGCG on different digestive tract cancer cell lines can find target sites of its anti-neoplastic effect and provide a theoretical basis for its clinical application in the treatment of cancers.

METHODSMethyl thiazolyl tetrazolium (MTT) analysis was made to detect the differential sensitivities of eight digestive tract cancer cell lines to EGCG. The effect of EGCG on cell cycle distribution of sensitive cancer cell line was measured by flow cytometry. By polymerase chain reaction (PCR)-enzyme linked immunosorbent assay (ELISA) protocol, the influence of EGCG on telomerase activity of sensitive cancer cell line was also investigated. RT-PCR method was employed to detect the influence of EGCG on the expressions of hTERT, c-myc, p53 and mad1 genes in sensitive cancer cell line.

RESULTSEGCG exhibited dose-dependent killing effects on all eight digestive tract cancer cell lines. The 50% inhibitory concentration (IC50) of SW1116, MKN45, BGC823, SGC7901, AGS, MKN28, HGC27 and LoVo cells were 51.7 micromol/L, 55.9 micromol/L, 68.5 micromol/L, 79.1 micromol/L, 83.8 micromol/L, 119.8 micromol/L, 183.2 micromol/L and 194.6 micromol/L, respectively. There were no apparent changes in cell cycle distribution of sensitive cancer cell line MKN45 48 hours after incubating with three different concentrations of EGCG compared with the controls. It was found that EGCG could suppress the telomerase activity of MKN45 cells, and the effects were dose- and time-dependent. After EGCG administration, the expression of hTERT and c-myc genes in MKN45 cells was decreased, that of the mad1 gene increased, and that of the p53 gene unchanged.

CONCLUSIONSEGCG has dose-dependent killing effects on different digestive tract cancer cell lines. Administration of EGCG has no obvious effect on cell cycle distribution of sensitive cancer cell line MKN45. The anti-neoplastic activity of EGCG might be due to the inhibition of telomerase activity by means of its influence on hTERT and the up-stream regulation genes.

Antineoplastic Agents ; pharmacology ; Catechin ; analogs & derivatives ; pharmacology ; Cell Cycle ; drug effects ; Cell Line, Tumor ; DNA-Binding Proteins ; genetics ; Digestive System Neoplasms ; drug therapy ; metabolism ; pathology ; Dose-Response Relationship, Drug ; Genes, myc ; Genes, p53 ; Humans ; Telomerase ; genetics ; metabolism

OBJECTIVETo explore the antitumoral effect of indirubin on androgen-independent prostate cancer PC-3 cells and its possible mechanisms.

METHODSWe measured the inhibitory effect of indirubin on the proliferation of prostate cancer PC-3 cells using MTT assay, detected their cell cycles by flow cytometry, and determined the expressions of the cell cycle regulatory protein cyclin D1 and its related downstream gene c-myc by Western blot.

RESULTSThe viability of the PC-3 cells was significantly decreased by indirubin in a concentration-dependent manner, reduced to 52. 2% and 13. 6% at 5 and 10 µmol/L, respectively. The cell cycle of the PC-3 cells was markedly inhibited by indirubin at 5 µmol/L, with the cells remarkably increased in the G0 and G1 phases and decreased in the S and G2/M phases. Meanwhile, indirubin also inhibited the expressions of cyclin D1 and c-myc in the Wnt signaling pathway.

CONCLUSIONIndirubin can suppress the proliferation of androgen-independent prostate cancer PC-3 cells, which may be associated with its inhibitory effect on the cell cycle and Wnt signaling pathway.

Antibiotics, Antineoplastic ; administration & dosage ; pharmacology ; Cell Cycle ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cell Survival ; drug effects ; Coloring Agents ; Cyclin D1 ; metabolism ; Dose-Response Relationship, Drug ; Genes, myc ; Humans ; Indoles ; administration & dosage ; pharmacology ; Male ; Prostatic Neoplasms, Castration-Resistant ; drug therapy ; pathology ; Proto-Oncogene Proteins c-myc ; metabolism ; Tetrazolium Salts ; Thiazoles

Dexamethasone converts pluripotent pancreatic AR42J cells into exocrine cells expressing digestive enzymes. In order to address molecular mechanism of this differentiation, we have investigated the role of mitogen-activated protein (MAP) kinase pathway and gene expressions of p21(waf1/cip1)and nuclear oncogenes (c-fos and c-myc) during AR42J cell differentiation. Dexamethasone markedly increased the intracellular and secreted amylase contents as well as its mRNA level. However, cell growth and DNA content were significantly decreased. With these phenotypic changes, AR42J cells induced transient mRNA expression of p21(waf1/cip1)gene, which reached maximal level by 6 h and then declined gradually toward basal state. In contrast to p21(waf1/cip1), c-fos gene expression was transiently inhibited by 6 h and then recovered to basal level by 24 h. Increased c-myc expression detected after 3 h, peaked by 12 h, and remained elevated during the rest of observation. Dexamethasone inhibited epidermal growth factor-induced phosphorylation of extracellular signal regulated kinase. Inhibition of MAP kinase pathway by PD98059 resulted in further elevation of the dexamethasone-induced amylase mRNA and p21(waf1/cip1)gene expression. These results suggest that p21(waf1/cip1)and nuclear oncogenes are involved in dexamethasone-induced differentiation and inhibition of MAP kinase pathway accelerates the conversion of undifferentiated AR42J cells into amylase-secreting exocrine cells.
Amylases/genetics ; Animals ; Cell Differentiation/*drug effects ; Cell Division/drug effects ; Cell Line, Tumor ; Cyclins/genetics/*metabolism ; Dexamethasone/*pharmacology ; Gene Expression Regulation/drug effects ; Genes, fos/genetics ; Genes, myc/genetics ; MAP Kinase Signaling System/*drug effects ; Mitogen-Activated Protein Kinases/*metabolism ; Pancreas/cytology/*drug effects/enzymology/metabolism ; RNA, Messenger/genetics/metabolism ; Rats ; Support, Non-U.S. Gov't

OBJECTIVETo establish human bronchial epithelial cell lines over expressing oncogene and to investigate its application in detection of carcinogen-induced cell transformation.

METHODSMediated by retrovirus infection, human telomerase catalytic subunit, hTERT was introduced into immortal human bronchial epithelial cells (16HBE) and followed by introduction of the oncogenic allele H-Ras(V12), or c-Myc or empty vector, creating cell lines 16HBETR, 16HBETM and 16HBETV, respectively. Biological characteristics of these cell lines including morphology, proliferation, and chromosomal aberration were examined to access whether they were transformed. Soft agar experiment and nude mice subcutaneous injection were performed using pre-transformed 16HBE cells induced by known carcinogens, nickel sulfate (NiSO4) and 7, 8, -dihydrodiol-9, 10-epoxide benzo[a] pyrene (BPDE).

RESULTSWith detection of telomerase activity and Western blotting, the expression of target proteins was verified. Thus, the transgenic 16HBE cell lines were successfully established. Cells expressing oncogene H-Ras or c-Myc grew 30.3% or 10.4% faster than control cells. However, these cells failed to form colonies in soft agar or form tumor in nude mice. 16HBETR, 16HBETM cells obtained transformed phenotype at 5 wks, 11 wks, respectively after treatment with BPDE, which are 15 wks and 9 wks earlier than control cells 16HBETV (20 wks). Meanwhile, 16HBETR, 16HBETM cells obtained transformed phenotype at 11 wks, 14 wks, respectively after treatment with nickel sulfate, which are 21 wks and 18 wks earlier than control cells (32 wks).

CONCLUSIONWith the advantage of shorter latency, transgenic human cell transformation models could be used in potent carcinogen screening and applied to chemical-carcinogenesis mechanism study.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide ; toxicity ; Animals ; Carcinogenicity Tests ; Cell Line ; Cell Transformation, Neoplastic ; drug effects ; metabolism ; pathology ; Epithelial Cells ; Gene Expression ; Gene Expression Regulation ; Genes, myc ; Genes, ras ; Humans ; Mice ; Mice, Inbred BALB C ; Mice, Nude