The Effect of Inner Friction on Concrete Fracture Behavior under Biaxial Compression: A 3D Mesostructure Study.

The mechanical behavior of concrete under biaxial loading condition (especially biaxial compression) is one of the most important indexes to evaluate the quality of concrete. To study the mechanical behavior of concrete under biaxial compression at mesoscale, we adopted our recently developed 3D numerical model based on Voronoi tessellation and cohesive elements. A constitutive model considering the friction effect is used in the model to characterize the fracture behavior of all potential fracture surfaces inside the concrete. A series of numerical experiments with different biaxial compression stress ratios were carried out. It was found that with the increase of the biaxial compression ratio, the proportion of energy increment caused by friction stress increases. The effect of inner friction coefficient on the biaxial relative strength was also investigated, and this kind of study is hard to be carried out through laboratory experiments. The results show that the inner friction coefficient has a great influence on the biaxial relative strength of concrete, and there is a positive correlation between these two parameters. Based on the above rules, a conservative biaxial relative compression strength envelope is obtained by setting the inner friction coefficient as zero.

[Expression of MiR101 and EZH2 in Patients with Mantle Cell Lymphoma and Its Clinical Significance].

OBJECTIVE: To investigate the expression of miR-101 and EZH2 in patients with mantle cell lymphoma(MCL) and to analyze its correlation with clinical prognosis of MCL patients. METHODS: RQ-PCR and S-P immunohistochemistry were used to detect the expressions of miR-101 and EZH2 in tissue of MCL patients. CCK-8 was used to assay the effect of miR-100 minics on the proliferation of Jeko-1 and Mino cells; the flow cytometry with Annexin V/PI double staining was used to assay the apoptosis; Western blot was used to assay the effect of miR-101 minics on the expression of EZH2 protein in Jeko-1 and Mino cells. RESULTS: Compared with control group, miR-101 lowly expressed, and EZH2 protein highly expressed in MCL group, with very statistically significant difference(P<0.01).There was negative correlation between miR-101 and EZH2 expression(r=-0.638，P<0.05). The expression of miR-101 and EZH2 significantly correlated with B symptoms, International Prognostic Index(IPI) and Ann Arbor stage, respectively. Survival analysis showed that the overall survival(OS) rate of patients with low expression of miR-101 were significantly lower than that of patients with high miR-101 expression (P=0.0014), the OS rate of patients with EZH2 high expression were significantly lower than that of patients with EZH2 low expression (P=0.0093). The miR-100 minics could inhibit the proliferation of Jeko-1 and Mino cells, and increase the apoptotic rate. The expression of EZH2 protein was markedly suppressed by the miR-100 minics. CONCLUSION: The expression of miR-101 and EZH2 is different in MCL patients with different clinical stage and prognosis. The miR-101 can inhibit the cell proliferation and induce cell apoptosis of mantle cell lymphoma by targeting EZH2.

[Effects of Silencing NSD2 Gene by shRNA on Proliferation, Apoptosis and Akt /mTOR Signal Pathway in OCI-Ly3 Cells].

OBJECTIVE: To investigate the effect of silencing NSD2 gene by RNA interference on the proliferation, apoptosis and the alteration of Akt /mTOR signaling pathway in diffuse large B cell lymphoma OCI-Ly3 cells. METHODS: The shRNA targeting NSD2 gene was transfected into OCI-Ly3 cells by lentivirus infection. The NSD2 mRNA and protein were detected by real time Q-PCR and Western blot, respectively. The cell proliferation was detected by CCK-8 and apoptosis was measured by flow cytometry. The expressions of BCL-2, BAX, caspase-3, Akt, p-Akt, p-mTOR, p-P70S6K, H3K36me2 were detected by Western blot. RESULTS: After transfecting the OCI-Ly3 cells by NSD2-shRNA for 72 h, the expressions of NSD2 mRNA and protein both were down-regulated(P<0.05), the proliferation rate of cells in NSD2 shRNA group was significantly lower than that in control and Neg shRNA groups (P<0.05); the apoptosis rate of cells in NSD2 shRNA group was significantly higher than that in control and neg-shRNA group (30.37±4.22)% vs 1.36±0.52 % and 2.17±1.43)%(P<0.05); the expressions of BAX and caspase-3 were up-regulated, while the expression of BCL-2 was down-regulated; the H3K36me2 level significantly decreased as compared with control group, no obvious decrease of the total protein level of AKT was found, but the expressions of p-Akt, p-mTOR and p-70S6K were down-regulated. CONCLUSION: The silencing NSD2 gene can inhibit the proliferation and induce the apoptosis of OCI-Ly3 cells, their mechanisms may relate with regulating the H3K36me2 level, specifically inhibiting the activivty of AKT/mTOR signal pathway.

[Effects of Sinomenine on Proliferation and Apoptosis of MCL Jeko-1 Cell Line and Its Mechanism].

OBJECTIVE: To investigate the effects of sinomenine on growth and apoptosis of MCL Jeko-1 cell line and its mechanism. METHODS: The proliferation rate of Jeko-1 cells treated by different doses of sinomenine was assayed by MTT method; and the cell apoptosis was detected by flow cytometry. The expressions of Cyclin D1, BCL-2, BAX, Caspase-3, Akt signaling pathway protein Akt, phosphorylated-Akt (p-Akt), and phosphorylated-mTOR (p-mTOR), phosphorylated- P70S6K(p-P70S6K) were determined by Western blot. RESULTS: The growth of Jeko-1 cell line was inhibited by Sinomenine. The apoptosis rates of Jeko-1 cells treated by 0, 1, 2, and 4 mmol /L of Sinomenine for 24 hours were (2.21±1.05) %, (11. 29±2.42)%, (18.79±2.84) %, (31.05±3.52) % respectively, and with very statistically significant difference(P<0.01). The expressions of p-Akt, p-mTOR, p-P70S6K were down-regulated, but total Akt expression was not changed. The expressions of cyclin D1 and BCL-2 were down-regulated, but that of BAX, and Caspase-3 were up-regulated. CONCLUSION: The sinomenine can inhibit Jeko-1 cell proliferation, which may be realized through down-regulating the phosphorylation level of p-Akt, p-mTOR, and p-P70S6K, thus inhibiting the Akt signaling pathway and promoting the cell apoptosis.

[Effect of Monoamine Oxidase Inhibitor Phenelzine on Proliferation of Mantle Cell Lymphoma and Its Mechanism].

OBJECTIVE: To investigate the effect of monoamine oxidase inhibitor phenelzine on in vitro growth and proliferation of mantle cell lymphoma Jeko-1 cells and its possible mechanism. METHODS: MTT assay was used to observe the cell proliferation and to draw a growth curve. The cell apoptosis was measured by flow cytometry. The expressions of apoptosis-related protein and Wnt signal pathway as well as the level of acetylation of histone were analyzed by Western blot. RESULTS: Phenelzine inhibited proliferation and promoted apoptosis of Jeko-1 cells in a dose-dependent way by increasing the expression of apoptosis related protein BAX, Caspase-3 and p21, while decreasing anti-apoptotic protein BCL-2. In addition, phenelzine could upregulate histone H3K4mel, H3K4me2 and histone acetylated H3, without affecting hitone H3K4me3. Moreover, phosphorylation of GSF-3ß, ß-catenin, c-myc and cyclinD1 decreased after exposure to phenelzine for 24 hours. CONCLUSION: Phenelzine can inhibit Jeko-1 cell proliferation and induce apoptosis by regulating methylation and acetylation of histone and suppressing Wnt/ß-catenin signal pathway, suggesting its therapeutic benefit for mantle cell lymophma.

Silencing of LSD1 gene modulates histone methylation and acetylation and induces the apoptosis of JeKo-1 and MOLT-4 cells.

Lysine-specific demethylase 1 (LSD1) has been identified and biochemically characterized in epigenetics; however, the pathological roles of its dysfunction in mantle cell lymphoma (MCL) and T-cell acute lymphoblastic leukemia remain to be elucidated. In this study, we evaluated LSD1, and histone H3 lysine 4 (H3K4)me1 and H3K4me2 expression in patients with MCL and silenced LSD1 in JeKo­1 and MOLT­4 cells, in order to define its role in JeKo­1 and MOLT­4 cell proliferation and apoptosis. We retrospectively analyzed the protein expression of LSD1, and mono- and dimethylated H3K4 (H3K4me1 and H3K4me2), and cyclin D1 and Ki67 in 30 cases of MCL by immunohistochemistry. The correlation of LSD1, H3K4me1 and H3K4me2 with Ki67 was determined by statistical analysis. LSD1 was silenced by small interfering RNA (siRNA). Cell apoptosis and cell proliferation were detected by flow cytometry and 3-(4,5-dimethylthiazol­2-yl)­2,5-diphenyltetrazolium bromide (MTT) assay. The protein expression levels of LSD1, histone methylated H3K4, histone acetylated H3, cyclin D1, apoptotic proteins, p15 and DNA methyltransferase 1 (DNMT1) were examined by western blot analysis. We demonstrated that LSD1 was upregulated, and that H3K4me1 and H3K4me2 were downregulated in the cases with MCL, compared to those with proliferative lymphadenitis (p<0.05). LSD1 positively correlated with Ki67 in MCL [Cohen's kappa (κ)=0.667, p<0.01]. There was no significant correlation between H3K4me1 and H3K4me2, and Ki67 (κ=-0.182, p>0.05, κ=-0.200, p>0.05). The silencing of LSD1 decreased the levels of the apoptosis-related proteins, Bcl-2, pro-caspase-3 and C-myc, and decreased those of DNMT1 and increased p15, and resulted in the loss of cell viability and the induction apoptosis. The silencing of LSD1 increased the expression of H3K4me1 and H3K4me2, and histone acetylated H3 in the JeKo­1 and MOLT­4 cells. LSD1 siRNA also decreased cyclin D1 expression in the JeKo­1 cells. On the whole, our findings demonstrate that the overexpression of LSD1 may be associated with the pathogenesis in MCL. We demonstrated that the silencing of LSD1 is capable of removing the mono- and dimethyl groups from H3K4, and upregulating the histone acetylation of H3 in JeKo­1 and MOLT­4 cells. The silencing of LSD1 inhibited cell growth and induced cell apoptosis. Of note, in JeKo­1 cells, the silencing of LSD1 decreased cyclin D1 expression, which is one of the genes that contribute to the pathogenesis of MCL. LSD1 may thus be a possible therapeutic target in MCL and acute lymphoblastic leukemia MOLT­4 cells.

Mechanism of G9a inhibitor BIX­01294 acting on U251 glioma cells.

The present study aimed to investigate the differential expression and clinical significance of histone methyltransferase G9a, histone H3K9me2 and histone H3K9me1 in human brain glioma and adjacent tissue samples. It also aimed to observe the effect and mechanism of BIX­01294, as an inhibitor of methyltransferase G9a, on the proliferation, apoptosis, methylation of H3K9 and H3K27, and the acetylation in U251 glioma cells in vitro. The differential expression of methyltransferase G9a, histone H3K9me2 and histone H3K9me1 in in human brain glioma and adjacent tissues were analyzed by immunohistochemistry, a growth curve of U251 cells following treatment with BIX­01294 was determined using the MTT assay. In addition, the apoptosis percentage of U251 cells was analyzed by TUNEL assay and the expression levels of apoptosis­associated proteins, including B­cell lymphoma 2 (Bcl­2), Bcl­2­associated X protein (Bax), caspase­9 and caspase­3, and the acetylation of histones, including H3K27me1, H3K27me2 and H3 in U251 were analyzed by western blot following BIX­01294 treatment. The positive rate of G9a in glioma tissues was 86% (43/50), which was significantly different from 42% (21/50) in adjacent tissues (P<0.01). The positive rate of H3K9me2 in glioma tissues was 82% (41/50), which was significantly different from 38% (19/50) in adjacent tissues (χ²=18.38; P<0.01). The expression of G9a and H3K9me2 were associated with the World Health Organization (WHO) glioma grade. The positive rate of H3K9me1 in glioma tissues was 54% (27/50) and 44% (22/50) in adjacent tissues, though this result was not significantly different (χ²=1.21, P>0.05). BIX­01294 inhibited the proliferation of U251, downregulated expression of Bcl­2, and upregulated expression of Bax, caspase­3 and caspase­9, and induced apoptosis of U251. BIX­01294 downregulated H3K9me1, H3K9me2, H3K27me1 and H3K27me2, however, it did not affect the acetylation of H3K9me3 and H3. High expression of G9a and H3K9me2 in glioma tissue samples was associated with the WHO grade, which indicated that G9a and H3K9me2 may promote generation and development of glioma. BIX­01294 inhibited proliferation and induced apoptosis of glioma cells, changes in methylation of H3K9 and H3K27 resulting in conformational changes of chromosome may be an underlying mechanism. BIX­01294 may be a potential novel therapeutic agent in the treatment of glioma.

[Rituximab plus Autologous Hemotopoietic Stem Cell Transplantation for The Treatment of CD5 Positive Diffuse Large B Cell Lymphoma with Autoimmune Hemolytic Anemia].

OBJECTIVE: To summarize the clinical features and therapy experience of a case of CD5 positive diffuse large B cell lymphoma (CD5+ DLBCL) with autoimmune hemolytic anemia (AIHA). METHODS: A 49-years old patient was investigated. The routine blood examination, bone marrow smear, Coombs test, serological test, chest CT, abdominal MR and immunochemistry etc were performed for this patient; and therapeutic effects of the chemotherapy regimen consisting of rituximab plus autologous hematopoietic stem cell transplantation (auto-HSCT) were observed. RESULTS: The cervical lymphnode biopsy confirmed CD5+ DLBCL; the severe anemia, reticulocyte increase, Coombs test positive, and erythroid hyperplasia in bone marrow all suggested the occurence of autoimmune hemolytic anemia (AIHA). After plasma exchange, immune suppression using methylprednisolone, blood transfusion, one course of chemotherapy with "R-CHOP-E", the symptoms of AIHA in patient disappeared. After a continuous treatment for 3 courses of "R-CHOP-E", the bone marrow infiltration appeared, which was assessed as "PD", then the treatment was changed to the "R-ESHAP" for 4 courses, the patient was reassessed as "CR". The patient subsequently underwent auto-HSCT, followed up for 6 months, patientis still "CR". CONCLUSION: The status of the CD5+ DLBCL patient with AIHA is severe, and the prognosis is poor. The curative effect of the chemotherapy regimen with rituximab plus auto-HSCT for this patien is well.

[Analysis of Immunophenotypes in 329 Patients with B-ALL at Different Ages].

OBJECTIVE: To investigate the immunophenotypes of B cell acute lymphoblastic leukemia (B-ALL) in patients at different age and to explore its clinical application in prognosis prediction and individualized treatment. METHOD: The immunophenotyping in 329 patients with B-ALL at different ages was performed by CD45/SSC gate four-color fluorescence flow cytometry. RESULTS: In all patients detected the highest incidence of lymphoid-associated antigens was CD19, HLA-DR, CyCD79a and cTdT, followed by CD10, CD22, CD34, CD38, CD20 and CyIg. B-ALL showed a higher concomitant expression rate of myeloid antigens CD13 and CD33; the CD11b, CD15, CD117 and T antigens (CD4, CD7 and CD56) were rarely expressed. CD10⁻ pro-B acute lymphoblastic leukemia (Pro - B-ALL) was predo-minant in infantile group (60%) with CD117 higher expression (40%). Subtype Pro-B-ALL was rarely expressed in childhood and adolescent group, but the incidence of disease increased as the age increase, the incidence of youth group (22.7%) and middle-aged' group (14.8%) were significantly higher than childhood group (4.4%). The influence of age on immunophenotypic characteristics of the adult B-ALL was not significant, the heterogeneity of antigen expression was less in the adult patients at different ages. The expression of CD10 and CD38 was lower, while expression of CD34, CD13 and CD33 were higher in adult patients than those in children patients. There was no significant difference in incidence of precursor-B-ALL (Pre-B-ALL) among different age groups (P > 0.05), but its incidence increased along with age increasing, and the expression of CD20 was higher in Pre - B-ALL than that in Pro - B-ALL and common B-ALL. CONCLUSION: The immunophenotype characteristics of B-ALL in the patients at different ages is of great value in prediction for disease prognosis and guidence of individualized treatment.

[Effects of Silencing AKT Gene by shRNA on Proliferation, Apoptosis and Notch1 Signal Pathway in Jeko-1 Cells].

OBJECTIVE: To investigate the effects of silencing AKT gene by RNA interference on the proliferation, apoptosis and the expression of Notch1 signal pathway-related proteins in mantle cell lymphoma Jeko-1 cell line. METHODS: The hairpin-like oligonucleotide sequences targeting AKT gene were designed and transfected into Jeko-1 cells by lipofectamine(TM) 2000. The AKT mRNA and protein were detected by RQ-PCR and Western blot respectively. Cell growth was determined by MTT. Cell apoptosis was analyzed by flow cytometry. The expression of BCL-2, BAX, caspase-3, caspase-9, p-AKT, Notch1, HES1 was detected by Western blot. RESULTS: AKT mRNA was markedly suppressed by the shRNA targeting AKT. AKT shRNA suppressed the proliferation of Jeko-1 cells and induced apoptosis of these cells. The cell apoptotic rates were (37.72±4.39)%, (2.62±1.53)%, (1.57±0.42)% in AKT shRNA, Neg-shRNA and Control, respectively, The difference between them was statistically significant (P<0.01). AKT shRNA down-regulated the expression of Bcl-2, AKT p-AKT , Notch1, and HES1, up-regulated the expression of BAX, caspase-3, caspase-9. CONCLUSION: Silencing AKT gene can inhibit the proliferation of Jeko-1 cells line, induc cell apoptosis, its mechanism may be associated with specially inhibiting PI3K/AKT signaling pathway and down-regulating the activity of Notch1 signaling pathway.

[Expression Changes of ß-catenin and P-GSK-3ß in Patients with Mantle Cell Lymphoma].

OBJECTIVE: This study was purposed to detect the expressions of ß-catenin and P-GSK-3 ß in Wnt signaling pathway of patients with mantle cell lymphoma(MCL), and investigate its relationship with the pathogenesis of MCL. METHODS: The expression levels of ß -catenin protein and P-GSK-3 protein in mantle cell lymphoma and hyperplastic lymphadenitis were detected by using anti-ß-catenin, P-GSK-3ß polyclonal antibody and S-P staining technique. RESULTS: The abnormal expression of ß-catenin protein(73.33%) in mantle cell lymphoma group was significantly higher than that (6.7%) in reactive lymph node hyperplasia group (P<0.05); and the positive rate of P-GSK-3 ß(66.67%) in mantle cell lymphoma group was significantly higher than that (16.67%) in reactive hyperplasia of lymph node group (P<0.05). Spearman correlation analysis showed that there was obvious positive correlation (R=0.852, P<0.01). CONCLUSION: The abnormal high expressions of ß-catenin and P-GSK-3 ß protein have been confirmed to appeare in mantle cell lymphoma.

[Antiproliferative Effect of Specific Inhibitor XAV939 for ß-catenin on MCL Jeko-1 Cells].

OBJECTIVE: To investigate the effect of short hairpin RNA (shRNA) and XAV939, a specific inhibitor for ß-catenin, on growth and apoptosis of mantle cell lymphoma(MCL) Jeko-1 cell line. METHODS: ß-catenin shRNA eukaryotic expression vector was transfected into Jeko-1 cells, the antiproliferative effect of shRNA on Jeko-1 cells was detected by RT-PCR and Western blot. The proliferation inhibitory rate of Jeko-1 cells treated by different doses of XAV939 was assayed by MTT method; the level of apoptosis of Jeko-1 cells was detected by flow cytometry; the expression levels of apoptosis-related protein BCL-2, BAX, CyclinD1, C-MYC and Caspase-3 in Jeko-1 cells were determined by Western blot. RESULTS: The expression of ß-catenin mRNA and growth of Jeko-1 cell line were inhibited by shRNA; after Jeko-1 cells treated with 0,2 and 8 µmol/L XAV939 for 48 hours, the cell proliferation rate decreased, while the cell apoptosis rate increased, the expressions of apoptosis-related protein BCL-2, CyclinD1 and C-MYC were down-regulated, on the contrary, the expression of BAX and caspase-3 were up-regulated. CONCLUSION: The specific inhibition of ß-catenin can inhibit Jeko-1 cell proliferation and promote the cell apoptosis.

[Effect of silencing NOTCH1 gene by shRNA interference on AKT/mTOR pathway in mantle cell lymphoma].

The study was purposed to investigate the effect of silencing NOTCH1 gene by shRNA interference on the proliferation, apoptosis and the expression of AKT signaling pathway-related proteins in mantle cell lymphoma Jeko-1 cell line. The hairpin-like oligonucleotide sequences targeting NOTCH1 gene were designed and transfected into Jeko-1 cells by lipofectamine (TM) 2 000. NOTCH1 mRNA and protein were detected respectively by RT-PCR and Western blot. Cell growth was determined by MTT. Cell apoptosis was analyzed by flow cytometry. The expressions of BCL-2, BAX, procaspase-3, procaspase-9, Akt, p-Akt, p-mTOR, p-P70S6K were detected by Western blot. The results showed that NOTCH1 mRNA expression was markedly suppressed by the shRNA targeting NOTCH1. NOTCH1 shRNA suppressed the proliferation of Jeko-1 cells and induced apoptosis of these cells. The cell apoptotic rate was (34.5 ± 3.4)%, (2.4 ± 1.3) %, (1.7 ± 0.6) % in NOTCH1 shRNA, Neg-shRNA and blank groups, respectively, and the difference between them was statistically significant (P < 0.01). NOTCH1 shRNA down-regulated the expression of BCL-2, procaspase-3, procaspase 9, p-Akt, p-mTOR and p-70S6K, up-regulated the expression of BAX, but no change protein expression of Akt was observed. It is concluded that the silencing NOTCH1 gene expression by shRNA interference may inhibit Jeko-1 proliferation, induce the cell apoptosis, and the mechanisms may be associated with the inhibition of Akt/mTOR signaling pathway by dephosphorylation.

[Application of surface-enhanced Raman spectroscopy to the determination of trace chemical hazards in food products].

In the present review, we summarized the research progress in applying SERS for the determination of illegal food additives, residual pesticides, banned or restricted antibiotics and other drugs. The nanosubstrates used in these studies included, but were not limited to, gold and silver nanosphere colloids, solid surface gold coated nanosubstrates, bimetallic nanosubstrates and spherical magnetic-core gold-shell nanoparticles, and etc. Standard solutions of a targeted chemical were normally tested first before analysis of relevant food in which the targeted chemical was commonly detected, and the tested food products included dairy products, condiments (such as chili powder and spices), fish, fruits and vegetables. The intensity of surface-enhanced Raman scattering signal is affected by various factors, which makes it difficult to obtain reproducible spectra. In addition, interferences of non-targeted food components on the target molecules during SERS analyses further makes it difficult to apply SERS as a routine analytic technique, despite its high specificity and sensitivity. Nevertheless, SERS is a new tool with great potential for analysis of trace amounts of chemical hazards in various food products and other complex systems.

[Antiproliferative effects of LY294002 on MCL Jeko-1 cell line and its mechanism].

This study was aimed to investigate the effects of LY294002, a specific inhibitor of phosphatidylinositol 3-kinase, on growth and apoptosis of MCL Jeko-1 cell line and its mechanism. The proliferation inhibitory rate of Jeko-1 cells treated by different doses of LY294002 was assayed by MTT method; the level of apoptosis of Jeko-1 cells was detected by flow cytometry; the expression level of apoptosis-related protein Cyclin D1, Bcl-2, procaspase-3 and PI3K/Akt signaling pathway protein phosphorylated-Akt (p-Akt), phosphorylated-TOR (p-mTOR), phosphorylated-P70S6K (p-P70S6K) phosphorylated-Akt (p-Akt) in Jeko-1 cells were determined by Western blot. The results showed that the growth of Jeko-1 cell line was inhibited by LY294002. The apoptosis rates of Jeko-1 cells treated with 0, 5, 10 and 20 µmol/L of LY294002 for 24 hours were (3.25 ± 1.27)%, (11.34 ± 2.35)%, (22.81 ± 2.74)%, (43.61 ± 3.48)% respectively, the difference between them was statistically significant (P < 0.01). Phosphorylation levels of PI3K/Akt signaling pathway protein p-Akt, p-mTOR, p-P70S6K decreased, the expression of apoptosis-related protein cyclin D1, Bcl-2, procaspase-3 was down-regulated.It is concluded that the LY294002 can inhibit Jeko-1 cell proliferation, which may be realized through down-regulating the phosphorylation level of p-Akt, p-mTOR, p-P70S6K, inhibiting the P13k/Akt signaling pathway, and promoting the cell apoptosis.

[Effect of downregulation the expression of HDAC1 on cells differentiation of HL-60 cells].

This study is to investigate the effect of downregulation histone deacetylases 1 (HDAC1) gene by the technology of RNA interference on the differentiation of HL-60 cells line. The optimal segment targeting HDAC1 gene was designed and transfected into HL-60 cells by Lipofectamine 2000. The HDAC1 mRNA and protein level were detected by RT-PCR and Western blotting. The morphologic change of HL-60 cells was detected by an optical microscope with Wright-Giemsa. Cell differentiation was tested by NBT reduction assay. Expression of CD13, CD33 and CD14 was measured by flow cytometry. The results indicated that HDAC1 mRNA and protein were markedly suppressed by the siRNA targeting HDAC1 in a concentration-dependent manner. HDAC1 siRNA promoted cell differentiation. HL-60 cells became more mature in morphology after transfected to HDAC1 siRNA at a concentration of 30-60 nmol x L(-1) for 24 h. NBT reduction ability of HDAC1 siRNA with 30 nmol x L(-1) was 0.31 +/- 0.09, compared with negative control (0.20 +/- 0.02) (t = -3.1, P < 0.01), and with 60 nmol x L(-1) was 0.25 +/- 0.02 in comparison with negative control (0.21 +/- 0.04) (t = -2.12, P < 0.05). But it has no change in HDAC1 siRNA > or = 120 nmol x L(-1). After transfection with 60 nmol x L(-1) HDAC1 siRNA to HL-60 cells, the expression of CD13 was (96.50 +/- 0.70)% in compared to siRNA-NC (3.39 +/- 0.68) % (t = 164.9, P < 0.000 5), CD33 was (66.73 +/- 0.50) % in compared to siRNA-NC (96.80 +/- 1.70) % (t = 43.4, P < 0.000 5). CD14 was (0.53 +/- 0.00) % by comparison with siRNA-NC (0.49 +/- 0.02) % (t = -0.97, P > 0.1). HDAC1 siRNA promoted cell differentiation in indicated concentration. HDAC1 might be one of the targets of gene therapy for leukemia.

[Effect of phenylhexyl isothiocyanate on Wnt/beta-catenin signaling pathway in Jurkat cell line].

This study was purposed to investigate the effect of phenylhexyl isothiocyanate (PHI) on Wnt/ß-catenin signaling pathway, histone acetylation, histone methylation and cell apoptosis in Jurkat cell line. The viability of Jurkat cells after treatment with PHI was tested by MTT. Apoptotic rate of Jurkat cells was measured by flow cytometry. The levels of Wnt/ß-catenin related proteins including ß-catenin, TCF, c-myc, and cyclinD1, histone acetylated H3 and H4, histone methylated H3K9 and H3K4 were detected by Western blot. The results showed that PHI inhibited the cell growth and induced apoptosis in Jurkat cells in time-and dose-dependent manners. Its IC50 at 48 h was about 20 µmol/L. Expression of histone acetylated H3, H4 and histone methylated H3k4 increased after exposure to PHI for 3 h, while histone methylated H3K9 decreased. Expression of ß-catenin was not changed after exposure to PHI for 3 h, but expression of ß-catenin, and its cell cycle-related genes such as TCF, c-myc and cyclinD1 decreased after exposure to PHI for 7 h. It is concluded that PHI regulates acetylation and methylation of histone, inhibits Wnt/ß-catenin signal pathway, and is able to induce apoptosis and inhibits growth of Jurkat cells.

[Effect of phenylhexyl isothiocyanate on drug-resistance to imatinib in K562/G01 cell line].

OBJECTIVE: To investigate the effect of phenylhexyl isothiocyanate (PHI) on the drug-resistance to imatinib in K562/G01 cell line and to elucidate its possible mechanisms. METHODS: MTT assay was employed to access K562/G01 cell growth inhibition after exposure to PHI and/or imatinib at different concentrations. Apoptotic rate of K562/G01 cells was measured by flow cytometry. The levels of P-gp, P210(bcr-abl) and p-P210(bcr-abl) protein were detected by Western blot. RESULTS: PHI inhibited proliferation and induced apoptosis of K562/G01 cells after treated with PHI alone for 24 h. PHI concentration increased from 0 to 40 µmol/L, the inhibitory rate of cell proliferation from 0 to (51.22 ± 1.41)%, and the apoptosis rate from (3.76 ± 1.46)% to (35.35 ± 3.70)%. Combination of 10, 20, 40 µmol/L PHI and various concentrations of imatinib, IC50 s of imatinib were (10.49 ± 1.24), (6.33 ± 1.42), and (0.85 ± 0.17) µmol/L, respectively. When K562/G01 cells treated with 20 µmol/L PHI combined with 10 and 20µmol/L imatinib for 24 hours, apoptosis rate were (43.62 ± 4.23)% and (55.41 ± 4.35)%, respectively, being significantly higher than that with imatinib or PHI alone. PHI concentrations increased from 0 to 40 µmol/L for 7 hours, the P210(bcr-abl)/ß-actin decreased from (0.944 ± 0.034) to (0.392 ± 0.025), and the p-P210(bcr-abl)/ß-actin decreased from (0.906 ± 0.019) to (0.361 ± 0.021), while the alteration of P-gp was not seen. CONCLUSIONS: PHI inhibits the proliferation and induces apoptosis of K562/G01 cell line. PHI has synergistic effect with imatinib. It partially reverses the drug-resistance to imatinib. The mechanism of reversal of drug resistance in K562/G01 cells might be by inhibiting P210(bcr-abl) and p-P210(bcr-abl).

[Experimental study of SUV39H1 gene specific siRNA in human leukemia cell line].

OBJECTIVE: To study the effects of suppressor of variegation 3-9 homolog 1 (SUV39H1) gene silencing by small interfering RNA (siRNA) on the proliferation, tumor suppressor gene p15 expression and histone modification in acute myeloid leukemia cell line KG-1 cells, and to explore novel therapeutic target of leukemia. METHODS: The SUV39H1 gene specific siRNA was synthesized in vitro and transfected into KG-1 cells by Lipofectamine(TM) 2000. The SUV39H1 mRNA and protein were detected by RT-PCR and Western blot. Cell growth affected by SUV39H1 siRNA was determined by MTS. The expressions of tumor suppressor gene p15, histone methylation of H3K9 and histone acetylation of H3, H3K9, H3K14, H3K27 and H4 were detected by Western blot. RESULTS: SUV39H1 mRNA was markedly suppressed by the SUV39H1 specific siRNA in a concentration-dependent manner. SUV39H1 siRNA inhibited the proliferation of KG-1 cells. Proliferation inhibition rate was (23.57 ± 1.98)%, (48.69 ± 1.84)%, (62.69 ± 1.61)% and (81.06 ± 3.22)% after transfected with SUV39H1 siRNA at 30, 60, 120 and 240 nmol/L for 48 hours, respectively. SUV39H1 siRNA down-regulated histone tri-methylated-H3K9 by 25%, 33% and 49% compared to control group when treated with SUV39H1 siRNA at 30, 60 and 120 nmol/L for 48 hours, while up-regulated histone acetylated H3K9 by 1.83, 2.16 and 3.07 folds, and global histone H3 in 1.35, 1.87 and 2.37 folds, but no changes were observed in histone acetylation of H3K14, H3K27 and H4. Expression of p15 increased 1.52, 2.89 and 3.08 folds after SUV39H1 siRNA treatment. CONCLUSIONS: SUV39H1 gene silencing could induce the re-expression of p15 and inhibit cell proliferation by down-regulation of histone methylation of H3K9, up-regulation of histone acetylation of H3K9 and global H3. SUV39H1 might be a new target for cancer therapy.

[Effect of SUV39H1 siRNA silence on apoptosis and proliferation of acute myelogenous leukemia KG-1 cell line].

This study was aimed to investigate the effects of SUV39H1 siRNA on proliferation and apoptosis of acute myelogenous leukemia KG-1 cell line. The small interfering RNA (siRNA) targeting SUV39H1 gene was designed and transfected into KG-1 cells by Lipofectamine(TM) 2000. Cell growth affected by SUV39H1 siRNA was determined by MTS method. Cell apoptosis was measured by flow cytometry. The expressions of P15 and anti-apoptosis protein such as BCL-2, procaspase-9, procaspase-3 and C-MYC were detected by Western blot. The results indicated that siRNA targeting SUV39H1 inhibited proliferation of KG-1 cells. Proliferated rates were (76.43 ± 1.98)%, (51.31 ± 1.84)%, (37.31 ± 1.61)%, (18.94 ± 3.22)% respectively after transfection with SUV39H1 siRNA at 30, 60, 120, 240 nmol/L for 48 h, while P15 expression was upregulated. Apoptotic cells significantly increased, apoptotic rates were (40.2 ± 5.1)%, (56.8 ± 4.8)%, (71.6 ± 5.6)% respectively after transfection with siRNA targeting SUV39H1 at 30, 60, 120 nmol/L (P < 0.05). The protein expression of BCL-2, procaspase-9, procaspase-3, C-MYC was downregulated after transfection. It is concluded that the siRNA targeting SUV39H1 inhibits cell growth and induces cell apoptosis of KG-1 cell line, which may be a new therapeutic target in human leukemia.