Dimethyl sulfoxide (DMSO), a well-known differentiation inducer in several myeloid cells, induces G1 phase arrest in many cell lines. In this study, we investigated the possibility of using DMSO to arrest H18 hybridoma cells to the G1 phase and monitor whether the arrest improves antibody production. We showed that DMSO in concentration ranging between 0.3% and 0.6% efficiently arrested H18 hybridoma cells in G1 phase. In our experiment, > 80% of cells grown for 36h in presence of the 0.6% DMSO were arrested in G1. Furthermore, expression levels of P27 were up-regulated tow fold during the G1 phase. Higher concentration of DMSO at 0.9% leads to cytotoxicity. Herein we show a simple way, a two-stage process for antibody production, which consists of a proliferation phase leading to the desired cell density, followed by an extended production phase during which the cells remain at G1 phase. Our observation that the addition of DMSO results in increase antibody production is of significance in further use of hybridoma cells in high density large scale cell culture.
Animals ; Antibodies, Monoclonal ; biosynthesis ; Cell Proliferation ; drug effects ; Dimethyl Sulfoxide ; pharmacology ; G1 Phase ; drug effects ; Hybridomas ; drug effects ; immunology ; Mice ; Proliferating Cell Nuclear Antigen ; analysis

OBJECTIVETo study the influence of Benzo (a) pyrene on cell cycle distribution of quiescent diploid human embryonic lung fibroblast (HELF) cells.

METHODSHELF cells were synchronized at G0 phase of cell cycle by 0.5% serum starvation for 48 hours and identified by flow cytometry (FCM). Cells were treated with 20 micromol/L benzo (a) pyrene for 4 h and detected for the changes of cell cycle distribution 0 h, 24 h, 48 h after treatment respectively. HELF cells were treated with 0, 5, 10, 20 micromol/L Benzo (a) pyrene for 24 h and detected for cell cycle regulators p53, p21 and p16 expression changes using Western Blotting method. On the other hand, the dynamic changes of these regulators were also been detected within 24 h after 20 micromol/L Benzo (a) pyrene treated for 4h.

RESULTS0.5% Serum starvation for 48 hours could effectively synchronize HELF at G0 stage and G0 reached 78%. Well-modulated control cells entered into cell cycle to synthesize DNA and cells at S phase reached 43.9% 24 h after serum re-stimulate, while 20 micromol/L B (a) P treated cells were arrested in G1 stage. Control cells entered into the G1 stage of next cell cycle another 24 h later, B (a) P treated cells recovered from G1 arrest, 26.5% of which reached S phase, having a delay of almost 24 h compared with controls. After a series of B (a) P concentrations acting for 24 h, we found that P53 and P21 expression increased dramatically. On the other hand, P53 and P21 increased 4 h after treatment, P53 recovered to normal level after 12 h while P21 kept increasing in 24 h. P16 initially decreased and became normal at 24 h.

CONCLUSIONB (a) P induced quiescent HELF cells undergoing a reversible G1 arrest related with p53-p21 pathway.

Benzo(a)pyrene ; toxicity ; Carcinogens ; toxicity ; Cell Cycle ; drug effects ; Cells, Cultured ; Diploidy ; Fibroblasts ; cytology ; drug effects ; Flow Cytometry ; G1 Phase ; drug effects ; Humans ; Lung ; cytology ; embryology

To explore the hematopoiesis inhibition mechanisms of interferon-gamma (IFN-gamma), the effects of IFN-gamma on the expression of the cyclin D in the umbilical cord blood hematopoietic stem/progenitor cells were observed. In the experiments the CD34(+) cells were isolated from the cord blood with MIDI-MACS system; semi-solid methylcellulose culture technique was used to measure the formation of CFU-GM; the expression levels of cyclin D isoforms were assayed by semi-quantitative RT-PCR, after the hematopoietic stem/progenitor cells were incubated with IFN-gamma. The results indicated that IFN-gamma could inhibit the formation of CFU-GM and down-regulate the expression of cyclin D2 and cyclin D3 at the mRNA level. It is concluded that the IFN-gamma could inhibit the proliferation of hematopoietic stem cells and down-regulate the expression of cyclin D, that may be one mechanism underlying the hematopoietic inhibition of IFN-gamma.
Cyclin D ; Cyclins ; genetics ; Fetal Blood ; cytology ; G1 Phase ; Hematopoietic Stem Cells ; drug effects ; metabolism ; Humans ; Interferon-gamma ; pharmacology ; Protein Isoforms ; RNA, Messenger ; analysis

OBJECTIVETo investigate the effect of water extracts from Duhuo Jisheng decoction on chondrocyte G1 phase.

METHODSChondrocytes were collected from four-week-old SD rats to establish the chondrocyte in vitro culture system. The third generation of chondrocytes was intervened. MTT method was used to measure the effect of water extracts from different concentrations of Duhuo Jisheng decoction on chondrocyte activity. The expressions of Chondrocyte Cyclin D1, CDK4, CDK6 and P21 mRNA in the blank group and low, middle and high-dose groups (100, 200, 400 mg x L(-1)) were detected by RT-PCR method.

RESULTThe MTT assay showed that the chondrocyte activity significantly increased within specific drug concentrations (50-800 mg x L(-1)) (P < 0.01); After the intervention for 24 h, the expressions of CyclinD1, CDK4 and CDK6 mRNA in all dose groups notably increased (P < 0.05), with the maximum expressions at the concentration of 200 mg x L(-1); The expression of P21 mRNA decreased, particularly at the concentration of 200 mg x L(-1) (P < 0.01).

CONCLUSIONWater extracts from Duhuo Jisheng decoction can promote chondrocyte proliferation by effecting the expression of chondrocyte G1 phase regulator mRNA.

Animals ; Cell Cycle Proteins ; genetics ; metabolism ; Cell Proliferation ; drug effects ; Cells, Cultured ; Chondrocytes ; cytology ; drug effects ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; G1 Phase ; drug effects ; Gene Expression Regulation ; drug effects ; Male ; RNA, Messenger ; genetics ; metabolism ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo investigate the biological activity and molecular mechanism of interferon alpha (IFNalpha) on human myeloma cell line Sko-007.

METHODSThe effect of IFNalpha on the growth of Sko-007 cells was measured by MTT assay. Cells cycle distribution and the expression of two IL-6 receptor chains (IL-6R and gp130) on Sko-007 cell surface in the absence or presence of IFNalpha were monitored by FACS analysis. The activation state of protein kinase ERK, which is involved in Ras/MAPK signal transduction pathway mediating cell survival and proliferation, and the expression of anti-apoptotic Bcl-2 family proteins-Bcl-2, Bcl-x(L) and Mcl-1 in Sko-007 cells with or without IFNalpha were determined by immunoblot assay.

RESULTIFNalpha arrested Sko-007 cell cycle progression. After stimulation with IFNalpha, an obvious increase in G(0)/G(1) phase (41.1%-->84.1%) and decrease in S phase (57.1%-->13.3%) of Sko-007 cell cycle distribution can be observed. Moreover, the proliferation of Sko-007 cells was dramatically inhibited in the presence of IFNalpha, with a maximal inhibitory rate up to 88%. In addition, the expression of gp130 on cell surface, the activation of protein kinase ERK and the expression of Bcl-2 and Bcl-x(L) were all down-regualted in IFNalpha-stimulated Sko-007 cells.

CONCLUSIONThe inhibitory effect of IFNalpha on the proliferation of Sko-007 cells was mediated by gp130 down-regulation, degradation of Bcl-2 family anti-apoptotic proteins and inhibition of ERK activation.

Antigens, CD ; drug effects ; metabolism ; Cell Cycle ; drug effects ; Cell Division ; drug effects ; Cytokine Receptor gp130 ; Dose-Response Relationship, Drug ; Down-Regulation ; Enzyme Activation ; drug effects ; G1 Phase ; drug effects ; Humans ; Immunoblotting ; Interferon-alpha ; pharmacology ; Membrane Glycoproteins ; drug effects ; metabolism ; Mitogen-Activated Protein Kinases ; metabolism ; Multiple Myeloma ; metabolism ; pathology ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Receptors, Interleukin-6 ; drug effects ; metabolism ; Resting Phase, Cell Cycle ; drug effects ; S Phase ; drug effects ; Tumor Cells, Cultured ; drug effects ; metabolism ; bcl-X Protein

In order to investigate the anti-tumor activity of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors and the mechanism underlying the cell proliferation and apoptosis modulated in myeloma cells, the effects of mevastatin, an HMG-CoA reductase inhibitor, on cell growth, cell cycle progression and apoptosis in U266 human multiple myeloma (MM) cell line in vitro were explored by MTT colorimetric assay, morphologic observation, flow cytometry, DNA gel electrophoresis, and RT-PCR. The results demonstrated that mevastatin inhibited the growth of U266 cells in time- and dose-dependent manners. Cell cycle analysis showed that U266 cells underwent G(0)/G(1) arrest under exposure to mevastatin, but it did not affect p27 expression at both mRNA and protein level. Morphologic observations revealed cytoplasm shrinkage, nuclear condensation and fragmentation in mevastatin-treated cells, and fraction of annexin V(+)PI(-) cells increased significantly in the presence of the agent as determined by flow cytometric assay. In addition, mevastatin caused the collapse of mitochondrial transmembrane potential (Deltapsim), induced DNA fragmentation, and down-regulated the mRNA expression of bcl-2. The growth-inhibitory, cell cycle arresting, and proapoptotic effects of mevastatin in U266 cells could be effectively reversed by the addition of mevalonate (MVA), the immediate endproduct of the reaction catalyzed by HMG-CoA reductase. It is concluded that mevastatin suppresses proliferation by inducing G(0)/G(1) phase arrest and triggering apoptosis via down-regulation of bcl-2 and reduction of Deltapsim, which may be attributed to the inhibition of MVA pathway by mevastatin. Statins including mevastatin may find their future application in the treatment of MM.
Apoptosis ; drug effects ; Cell Division ; drug effects ; Cell Line, Tumor ; G1 Phase ; drug effects ; Genes, bcl-2 ; Humans ; Hydroxymethylglutaryl-CoA Reductase Inhibitors ; pharmacology ; Lovastatin ; analogs & derivatives ; pharmacology ; Multiple Myeloma ; drug therapy ; pathology

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 investigate the effect of curcumin on apoptosis of human prostatic stromal cells.

METHODDifferent concentrations of curcumin were added into culture media system to induce apoptosis of human prostatic stromal cells. The apoptosis was detected by MTT assay, flow cytometry, RT-PCR and TUNEL method.

RESULTCurcumin at concentrations of 10-40 micromol x L(-1) could inhibit the proliferation of human prostatic stromal cells in a doseand time-dependent manner (P < 0.05). Characteristic apoptosis were confirmed by TUNEL RT-PCR manifest downregulation of Bcl-2/Bax (P < 0.05). Cell cycle was arrested into G1 phase by curcumin.

CONCLUSIONCurcumin can induce apoptosis in human prostatic stromal cells by down-regulation of Bcl-2/Bax.

Apoptosis ; drug effects ; Curcumin ; pharmacology ; Flow Cytometry ; G1 Phase ; drug effects ; Humans ; In Situ Nick-End Labeling ; Male ; Prostate ; cytology ; Reverse Transcriptase Polymerase Chain Reaction ; Stromal Cells ; drug effects ; metabolism ; Tumor Cells, Cultured ; bcl-2-Associated X Protein ; metabolism

OBJECTIVETo explore the effects of epigallocatechin-3-gallate (EGCG) on the proliferation of human oral epithelial cancer cell line KB cells and the molecular mechanisms.

METHODKB cells were treated with various concentrations of EGCG for 24 or 48 h. MTT assay was used to test the cell viability. The changes of cell cycle in KB cells treated with EGCG for 48 h were analyzed using flow cytometry. The expressions of cyclin A, cyclin D1 and cyclin E were detected by RT-PCR and Western blotting.

RESULTThe viability of KB cells treated with various concentrations of EGCG (25, 50, 100, 200, 400, and 800 micromol/L) for 48 h were decreased to (85.4-/+2.4)%, (80.4-/+2.8)%, (51.5-/+4.5)%, (30.2-/+1.9)%, (25.3-/+1.5)%, (20.0-/+1.1)%, respectively, showing significant difference from that of the control group [(100.0-/+2.2)%, P<0.05). EGCG decreased the viabilities of KB cells in a dose-dependent manner. Flow cytometry demonstrated that treatment with EGCG significantly increased the cell percentage in sub-G1 phase, which was (73.5-/+4.4)% after a 48-h EGCG treatment, significantly different from that in the control group [(47.3-/+3.5)%, P<0.05). EGCG-induced G1 phase arrest was correlated to the down-regulation of cyclin A and cyclin E.

CONCLUSIONEGCG inhibits the proliferation of KB cells by inducing G1 phase arrest, which involves the downregulation of cyclin E.

Catechin ; analogs & derivatives ; pharmacology ; Cell Cycle ; drug effects ; Cell Proliferation ; drug effects ; Cyclin E ; metabolism ; Flow Cytometry ; G1 Phase Cell Cycle Checkpoints ; drug effects ; Humans ; KB Cells ; Oncogene Proteins ; metabolism

OBJECTIVETo explore the effect of p21(WAF1) on the proliferation and the sensitivity to Vp16 of leukemia cell line K562.

METHODSA p21(WAF1) retroviral expression vector pLXSN-p21(WAF1) was constructed by FuGENE 6, pLXSN-p21(WAF1) and pLXSN-neo, and transfected into p21(WAF1) defect leukemia cell line K562. After selected with G418, K562-p21(WAF1) cell clones that stably expressed p21(WAF1) were isolated. The ectopic expressions of p21(WAF1) mRNA and protein in K562-p21(WAF1) were identified by RT-PCR and Western b1ot. The cell growth rate was tested by trypan blue dye, the cell cycle by FCM and the sensitivity to Vpl6 by cell count and MTT assay.

RESULTSThe expression of p21(WAF1) protein and mRNA could be detected in K562-p21(WAF1) cells. A strong inhibition of cell proliferation was observed in K562-p21(WAF1) cell as compared with that of the control. The cell number in G(0)/G(1) phase was remarkably increased. The sensitivity to Vpl6 decreased, the IC (50) of K562-neo cells was (56.4 +/- 6.5) microgram/ml, and that of K562-p21(WAF1) cells was (131.0 +/- 8.7) microgram/ml (P < 0.01).

CONCLUSIONp21(WAF1) can inhibit the proliferation of leukemia cell and decrease its sensitivity to Vp16.

Antineoplastic Agents, Phytogenic ; pharmacology ; Blotting, Western ; Cell Division ; drug effects ; Cell Survival ; drug effects ; Cyclin-Dependent Kinase Inhibitor p21 ; Cyclins ; genetics ; metabolism ; physiology ; Dose-Response Relationship, Drug ; Etoposide ; pharmacology ; G1 Phase ; drug effects ; Gene Expression ; Humans ; K562 Cells ; cytology ; drug effects ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Resting Phase, Cell Cycle ; drug effects ; Reverse Transcriptase Polymerase Chain Reaction ; Time Factors