OBJECTIVETo study the antitumour effects of sodium valproate (VPA) on the proliferation, differentiation and cell cycle of Molt-4 cell and to investigate its demethylation mechanisms.

METHODSAfter Molt-4 cells trated with VPA at different concentrations, cell viability and growth curve were assessed by MTT assay. Cell cycle changes were analyzed by flow cytometry. The expression level of p15, DNA methyltransferase 1 (DNMT-1), DNMT3A and 3B mRNA were detected by RT-PCR and the methylation level was detected by hn-MSPCR.

RESULTSVPA significantly inhibited the proliferation of Molt-4 cells. After 48 h culture with 5.0 mmol/L VPA, the percentages of Molt-4 cells in G(0)/G(1) phase was (66.87 ± 3.31)% and in S phase was (8.47 ± 2.56)%, while in control group, the cells in G(0)/G(1) phase increased and in S phase decreased significantly. The p15 gene in Molt-4 cells failed to express due to its hypermethylation. The expression level of p15 gene mRNA increased significantly after exposure to VPA for 48 h. As compared with control group, the expression of DNMT-1 was down-regulated in a dose-dependent manner. The expression level of DNMT3B decreased at 10.0 mmol/L concentration.

CONCLUSIONVPA has a demethylation effect on p15 INK4B gene by inhibiting the DNMT-1 and DNMT3B gene activities to recover p15 gene activity, which arrests Molt-4 cell in G(0)/G(1) phase.

Cell Cycle ; drug effects ; Cell Line, Tumor ; DNA Methylation ; drug effects ; RNA, Messenger ; genetics ; Valproic Acid ; pharmacology

OBJECTIVETo construct a eukaryotic expression vector containing aldehyde dehydrogenase-2 (ALDH2) gene and investigate the effects and its possible mechanisms of ALDH2 gene on cell proliferation and anti-oxidative damage in the K562 cells.

METHODSAn eukaryotic expression vector containing the ALDH2 gene cloned from human hepatocytes was constructed and transfected into K562 cells by liposome. RT-PCR and Western blot were used to evaluate the expression of ALDH2. MTT assay was used to check the cell proliferation and trypan blue exclusion to check K562 cells damage induced by hydrogen peroxide (H2O2). RT-PCR and fluorescence spectrophotometry were used to determine the expression of heme oxygenase-1 (HO-1) and the generation of intracellular reactive oxygen species (ROS) respectively.

RESULTSRT-PCR and Western blot analysis showed distinct higher ALDH2 protein expression in gene transfected group. The latter group had a higher cell proliferation (P < 0.05) and survival rate against H2O2 induced-oxidative damage, being increased by 7.8 times (IC(50) was 12.3 µmol/L and 1.4 µmol/L for K562-pcDNA3.1-ALDH2 and control cells, respectively, P < 0.01). The HO-1 mRNA expression and the generation of intracellular ROS were downregulated at a specific concentration of H2O2 in the ALDH2 gene transfected group.

CONCLUSIONALDH2 gene transfection can protect K562 cells against oxidative damage, and the downregulation of HO-1 expression and intracellular ROS may be involved in this process.

Aldehyde Dehydrogenase ; Apoptosis ; drug effects ; Cell Proliferation ; drug effects ; Humans ; Hydrogen Peroxide ; K562 Cells ; RNA, Messenger ; genetics ; Transfection

OBJECTIVETo evaluate the biological effects of nano-hydroxyapatite/polyamide 66(nHA-PA66) on the growth and activity of osteoblast.

METHODSMTT assay was used to determine the growth of osteoblast, enzymatic measure was used to determine the activity of ALP and quantitative RT-PCR (QRT-PCR) to evaluate the changes of osteoclacin mRNA expression in osteoblasts treated by DMEM eluate of nHA-PA66.

RESULTSOsteoblasts of different test groups demonstrated relative proliferation rate ranging from 98% - 106% without dose-dependent effect. The ALP activity and osteocalcin mRNA expression were similar in test and control groups (P > 0.05).

CONCLUSIONnHA-PA66 has no negative effects on the osteoblast and its osteoblast-compatibility is proved.

Durapatite ; pharmacology ; Nylons ; pharmacology ; Osteoblasts ; drug effects ; Osteocalcin ; metabolism ; RNA, Messenger ; metabolism

OBJECTIVETo study methylation of Id4 gene and demethylation effect of arsenic trioxide (ATO) in Raji cells.

METHODSHuman Burkitt's Raji lymphoma cells were cultared and treated with ATO at different concentrations and different time points. Methylated degree of Id4 gene was detected by methylation specificity polymerase chain reaction (MS-PCR), Id4 mRNA expression in Raji cell by reverse transcription polymerase chain reaction (RT-PCR), the growth of cell by MTT assay, and cell apoptosis and cycle distribution by Flow Cytometry (FCM).

RESULTS(1) The Id4 gene exhaustive methylation in control group, and hypermethylation in experimental group were reversed by ATO in a dose-dependent manner. (2) Id4 mRNA expression in Raji cells treated with ATO for 48 h increased gradually with ATO concentration increasing in experimental group. (3) Raji cell growth inhibited rates after different concentrations of ATO treatment for 24, 48, 72 h were 12.15% ∼ 92.17% in the experimental group (P < 0.05). (4) Apoptosis peak emerged after ATO treatment for 48 hours in experimental group, while a much lower apoptosis in control group. (5) After ATO treatment for 48 h in experiment group, the cells were arrested at G(0)/G(1) phase in a dose-dependent manner.

CONCLUSIONId4 gene presents exhaustive methylation in Raji cells. ATO can reverse the hypermethylation of Id4 gene and recover the expression of Id4 mRNA. Hypermethylation of Id4 gene is one of the reasons of Raji cells malignant proliferations.

Apoptosis ; drug effects ; Burkitt Lymphoma ; genetics ; Cell Line, Tumor ; DNA Methylation ; Humans ; RNA, Messenger ; genetics

OBJECTIVETo detect the expression changes of the demethylase TETs (Ten-eleven translocation enzymes) in human embryonic kidney cell (HEK293) exposed to high dose cadmium chloride (CdCl2), and to investigate the regulation effects of TETs on global genomic methylation.

METHODSHEK293 cells were exposed to CdCl2 for 24 h, 48 h and 72 h, the survival rate was tested by CCK-8 (cell counting kit-8) method, and the cell morphology was observed. The levels of TETs mRNA and protein were detected by fluorescence quantitative PCR and Western blot, respectively. The genomic DNA methylation level was detectedby pyro sequencing assay.

RESULTSCdCl2 had toxic effects on HEK293 cells, and the half inhibitory concentration (IC50) was 1.78 µmol/L. After exposure of CdCl2 for 24 h, 48 h and 72 h, the morphology of HEK293 cells was altered, and the high dose group (2.0 µmol/L) showed vacuolar changes and fuzzy appearance. The level of TET1 mRNA in groups of 0.0, 0.5, 1.0, 2.0 µmol/L were 0.23 ± 0.13, 0.48 ± 0.12, 0.59 ± 0.16 and 0.95 ± 0.39, respectively (F = 182.89, P = 0.002); The level of TET2 mRNA in groups of 0.0, 0.5, 1.0, 2.0 µmol/L were 0.23 ± 0.12, 0.32 ± 0.02,0.31 ± 0.10 and 0.34 ± 0.07, respectively (F = 27.94, P < 0.001); The level of TET3 mRNA in groups of 0.0, 0.5, 1.0, 2.0 µmol/L were 0.26 ± 0.10, 0.27 ± 0.11, 0.25 ± 0.11 and 0.28 ± 0.09, respectively (F = 1.76, P = 0.036). The interaction effect existed between exposure time and doses of TET1 mRNA, TET2 mRNA and TET3 mRNA (F values were 32.94, 23.04 and 13.78, respectively; P values were < 0.001, 0.041 and < 0.001, respectively). Western blot showed that in different exposure time and dose, the protein expression levels of TETs had the similar trend as mRNA levels. In 24 h (55.01 ± 3.62)%, 48 h (48.31 ± 8.99)%, 72 h (48.76 ± 6.60)%, the DNA methylation had significant differences (F = 18.50, P < 0.001); In groups of 0.0 µmol/L (55.29 ± 2.83)%, 0.5 µmol/L (55.35 ± 3.11)%, 1.0 µmol/L (48.58 ± 6.40)% and 2.0 µmol/L (43.56 ± 7.89)%, the differences of DNA methylation had significant differences (F = 7.03, P = 0.048); the effect of interaction was also existed (F = 2.73, P = 0.043).

CONCLUSIONIn the short term exposure to CdCl2, the levels of TETs mRNA and protein showed a trend of increase according to the exposure time and dose, and the methylation level of whole genomic DNA was also altered. The demethylase TETs may play a role in regulating the genomic methylation level of HEK293 exposed to cadmium.

Cadmium Chloride ; toxicity ; DNA Methylation ; Dioxygenases ; genetics ; Epithelial Cells ; drug effects ; HEK293 Cells ; Humans ; RNA, Messenger

OBJECTIVETo investigate the effects of small interfering RNA (siRNA) targeting YAP on the proliferation and apoptosis of human periodontal ligament stem cells (hPDLSCs).

METHODSSynthesized sequences of siRNA were transfected into hPDLSCs by Lipofectamine™ 2000. The expression of YAP was identified by using real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis. Proliferation activity was detected by using cell counting kit-8 (CCK-8). Changes in the cell cycle and apoptosis rate were detected by using flow cytometry. Results were analyzed by using SPSS 19.0, and P < 0.05 was considered statistically significant.

RESULTSExpression of YAP mRNA and protein were significantly downregulated after 48 h of transfection (P < 0.001). No obvious difference was found in the expression levels of YAP protein between 48 and 72 h, thus indicating that siRNA could inhibit the expression of YAP persistently and effectively. Proliferation activity was inhibited, and apoptosis rate was increased. Cell cycle was changed as the proportion of G₁and S phases increased (P < 0.01) and G₂ phase decreased (P < 0.05).

CONCLUSIONKnocking down YAP gene by siRNA could inhibit proliferation activity, induce apoptosis, and change the cell cycle of hPDLSCs. Thus, YAP could regulate the proliferation and apoptosis of hPDLSCs.

Adaptor Proteins, Signal Transducing ; Apoptosis ; drug effects ; Cell Cycle ; drug effects ; Cell Proliferation ; drug effects ; Down-Regulation ; Humans ; Periodontal Ligament ; drug effects ; Phosphoproteins ; RNA, Messenger ; RNA, Small Interfering ; pharmacology ; Stem Cells ; drug effects ; Transfection

OBJECTIVETo study the effects of licorice on the proliferation of intestinal crypt stem cell line IEC-6 and the expression of p53.

METHODSInduced by difluoro-methylornithine (DFMO), polyamine-depleted IEC-6 cells under growth inhibition were used as the pathological cell model in this study. Cells were divided into four groups, i. e., the control group, the DFMO-treated group, the high dose licorice group, and the low dose licorice group. The control group consisted of IEC-6 cells cultured in normal condition. The other three groups were all treated with 5 mmol/L DFMO. The high dose and low dose licorice groups were supplemented with 40 and 80 microg/mL licorice granule respectively. All the groups were cultured for 6 successive days. The cell number and viability were determined using flow cytometry. The level of p53 protein was detected by Western blot. The p53 mRNA levels and stability were detected using fluorescent quantitative Real-time PCR.

RESULTSCompared with the control group, the cell growth of the DFMO group was obviously inhibited on the 4th day (P < 0.05). The cell number increased more obviously in the low dose licorice and the high dose licorice groups in a dose-dependent way on the 6th day when compared with the DFMO group (P < 0.05). When compared with the control group, significantly elevated expression levels of p53 protein and mRNA in cells of the DFMO group were detected after 6-day treatment (P < 0.05). When compared with the DFMO group, the expression levels of p53 protein and mRNA were significantly down-regulated in the low dose licorice and the high dose licorice groups (P < 0.05). The degradation of p53 mRNA was the fastest in the control group, while the degradation speed of cells in the DFMO group was the slowest.

CONCLUSIONOne of mechanisms for protective and healing effects of licorice on the intestinal mucosa was possibly through down-regulating the stability of p53 mRNA, lowering the expression of p53, thus promoting the proliferation of the intestinal crypt stem cells.

Animals ; Cell Line ; Cell Proliferation ; Glycyrrhiza ; Intestines ; cytology ; metabolism ; RNA Stability ; drug effects ; RNA, Messenger ; genetics ; Rats ; Stem Cells ; cytology ; drug effects ; metabolism ; Tumor Suppressor Protein p53 ; metabolism

In order to investigate the potential role of human ERMAP gene in erythropoiesis, the ERMAP-dsDNA was designed, ERMAP-shRNA expressing plasmids was constructed, and ERMAP-shRNA/K562 cell was established. Cell morphology, biphenylamine staining, expression of cell surface antigens as well as quantitative level of human ERMAP gene were observed during K562 cells differentiating toward erythroid lineage induced by Ara-C. The results showed that at 72 hours after Ara-C treatment, ERMAP-shRNA/K562 cell size became large with increasing cytoplasm content. The percentage of biphenylamine positive cells increased from 1.17% to 2.04% (p < 0.05), but still lower than that in group K562 + Ara-C. The percentage of CD36(-)/CD235a(+) increased from 8.83% to 11.28%, CD36(+)/CD235a(+) increased from 1.23% to 2.64%, and CD36(+)/CD235a(-) increased from 0.59% to 1.47% respectively, which were all lower than that in group K562 + Ara-C at either time point. At the same time, the level of ERMAP expression increased slowly from 2.52 x 10(-3) to 4.53 x 10(-3), which was also significantly lower than that of group K562 + Ara-C. It is concluded that the ERMAP-shRNA inhibits the Ara-C-induced erythroid differentiation of K562 cells, which further suggests that there is relationship between hERMAP and erythroid differentiation and development.
Blood Group Antigens ; genetics ; Butyrophilins ; Cell Differentiation ; drug effects ; Cytarabine ; pharmacology ; Erythropoiesis ; drug effects ; Humans ; K562 Cells ; RNA, Messenger ; RNA, Small Interfering ; pharmacology

OBJECTIVESTo investigate whether antisense human telomerase reverse transcriptase (hTERT) could inhibit the activity of telomerase and the proliferation of K562 cells.

METHODSThe antisense plasmid was constructed by reverse insertion of hTERT PCR product into plasmid pLNCX-neo. Then the constructed plasmid was introduced into K562 cells by liposomes-mediated DNA transfection. The inhibition effects of telomerase on the proliferation of K562 cells were analyzed by MTT and colony formation assay, the telomerase activity of K562 cells by TRAP-PCR ELISA methods.

RESULTSThe growth rate of antisense hTERT transfected K562 cells was significantly lower than those of the controls, and the colony formation capacity of the transfected cells decreased significantly (P < 0.01), the colony number is (100.33 +/- 7.57)/10(3) cells, (92.67 +/- 5.86)/10(3) cells and (50.33 +/- 6.11)/10(3) cells for control K562 cells, K562 neo cells and antisense hTERT transfected HL60 cells, respectively. The telomerase activity of antisense hTERT transfected K562 cells was significantly inhibited.

CONCLUSIONThe expression of an antisense sequence to the mRNA sequence of telomerase protein subunit can inhibit the activity of telomerase, slow the cell growth and inhibit the capacity of colony formation of K562 cells.

Cell Division ; drug effects ; Humans ; K562 Cells ; Plasmids ; genetics ; RNA, Antisense ; genetics ; pharmacology ; RNA, Messenger ; genetics ; Telomerase ; drug effects ; genetics ; metabolism ; Transfection

OBJECTIVETo observe the effect of chronic lead contaminant on mRNA expression of protein kinase C (PKC) and calmodulin (CaM) in hippocampus of baby rats.

METHODSThe Wistar pregnant rats were randomly divided into 3 groups fed with distilled water and lead contained water (0.2% and 1.0% lead acetate) respectively. The lead exposure period was from the 0 day of pregnancy to the day when the offspring weaned. Then the baby rats were fed with lead water the same as their mothers. The cliff avoidance reflex within postnatal day 8 and step down test at postnatal day 50 were performed. Then pups were killed at postnatal day 8 and 50 respectively. Atomic absorption spectrometry was used to determine lead content of rats' brain. RT-PCR was used to observe mRNA expression of PKC and CaM in hippocampus of baby rats.

RESULTSThe brain lead content of test groups were much higher than that of the control group. The completion rate of cliff avoidance reflex and the score of step down test of test groups were lower than those in the control group (P < 0.05). Compared with control group, PKC and CaM mRNA expression of chronic lead exposure baby rats in the hippocampus had the down trend (P < 0.05).

CONCLUSIONThe decrease of PKC and CaM mRNA expression level in hippocampus has a great link with the impairment of learning and memory induced by lead in baby rats, which might be one of the molecule mechanisms of lead induced impairment of learning and memory.

Animals ; Calmodulin ; genetics ; metabolism ; Female ; Hippocampus ; drug effects ; metabolism ; Lead ; toxicity ; Learning ; drug effects ; Male ; Memory ; drug effects ; Protein Kinase C ; genetics ; metabolism ; RNA, Messenger ; genetics ; Rats