Effect of γδ T cells on the Proliferation, Apoptosis and Autophagy of Multiple Myeloma Cells / 中国实验血液学杂志

AbstractObjective: To investigate the effect of γδ T cells on the proliferation, apoptosis and autophagy of multiple myeloma cells.@*METHODS@#Peripheral blood mononuclear cells (PBMNC) were isolated from healthy volunteers, and stimulated with zoledronic acid (Zol) in combination with rhIL-2. Flow cytometry analysis was used to detected the purity of γδ T cells. γδ T cells were collected and co-cultured with RPMI-8226 or U-266 cells at different effector target ratios. The proliferation of RPMI-8226 or U-266 cell lines were detected by CCK-8. Cell cycle and cell apoptosis were detected by flow cytometry and Western blot．The expressions of autophagy-related proteins were detected by Western blot.@*RESULTS@#γδ T cells can be expanded in vitro. γδ T cells could inhibit the proliferation of RPMI-8226 or U-266 cells, induced cell cycle arrest and promoted apoptosis in an effector target-dependent manner. In addition, γδ T cells could induce autophagy of myeloma cells, inhibited the expression of autophagy-related PI3K, P-AKT and P-mTOR, while increased the expression of AMPK and Beclin-1.@*CONCLUSION@#γδ T cells can inhibit the proliferation of RPMI-8226 and U-266 myeloma cells, induce cell cycle arrest, promote apoptosis, and enhance autophagy in vitro. The mechanism may be related to inhibition of PI3K/AKT/mTOR signaling pathway and/or activation of AMPK/Beclin-1 signaling pathway.

Genotype Analysis of Pregnant Women with α- and β- Thalassemia in Fuzhou Area of Fujian Province in China / 中国实验血液学杂志

OBJECTIVE@#To analyze the genotype of pregnant women with α- and β- thalassemia in Fuzhou area of Fujian province in China.@*METHODS@#Blood routine examination and hemoglobin electrophoresis were performed for pregnant women, and positive samples were examined by gap polymerase chain reaction and reverse dot blot hybridization.@*RESULTS@#412 cases were diagnosed as α-thalassemia (63.9%); 201 cases were diagnosed as β-thalassemia (31.2%); 32 cases were diagnosed as α and β-composite thalassemia. There were 12 genotypes in α-thalassemia, whose major genotypes were --/αα, α/αα, -α/αα and αα/αα, with carrying rate of 64.32%, 20.14%, 7.77% and 1.94%, respectively. There were 10 genotypes in β- thalassemia, whose major genotypes were CD41-42/N, CD17/N, IVS-II-654/N and -28/N, with carrying rate of 30.84%, 27.86%, 15.92% and 10.45%, respectively. There were 9 genotypes in α and β-composite thalassemia, whose major genotypes were --/αα composited CD41-42/N, -α/αα composited CD41-42/N, --/αα composited CD17/N, with carrying rate of 18.75%, 15.62%, 15.62% respectively.@*CONCLUSION@#The major genotypes of pregnant women with α- and β- thalassemia in Fuzhou area of Fujian province in China are --/αα, α/αα, CD41-42/N and CD17/N. Thalassemia screening and prenatal gene diagnosis should be strengthened in Fuzhou area of Fujian province in China.

Effects of Autophagy Regulating Drugs on Proliferation, Apoptosis and Autophagy of Multiple Myeloma Cells / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate the effects of autophagy activator (rapamycin, RAPA) and autophagy inhibitor (hydroxychloroquine, HCQ and 3-methyl adenine, 3-MA) on the proliferation, apoptosis and autophagy of multiple myeloma cell line of RPMI8226.</p><p><b>METHODS</b>RPMI8226 cells were treated with autophagy regulating drugs of different concentrations. The proliferation and apoptosis of cells were determined by CCK-8 and flow cytometry, respectively. The expressions of apoptosis-related proteins BCL-2, caspase-3 and PARP protein were assessed by Western blot. Autophagy was detected by monodansylcadaverine staining. Autophagic protein (LC-3b) and apoptosis-related proteins (caspase-3, PARP and BCL-2) were analyzed by Western blot.</p><p><b>RESULTS</b>RAPA and HCQ inhibited the proliferation of RPMI8226 in a concentration- and time-dependent manner, and increased the apoptosis. However, 3-MA did not show significantly inhibitory effect on the proliferation and apoptosis of RPMI8226. MDC staining showed that the more autophagic vacuoles could be detected in the higher concentration of RAPA, but the less autophagic vacuoles in the higher concentration of HCQ and 3-MA. Western blot showed that RAPA increased the expression of LC3-II/LC3-I, caspase-3 and PARP, but inhibited the expression of BCL-2. HCQ inhibited the expression of LC3-II/LC3-I and BCL-2, but increased the expression of caspase-3 and PARP. 3-MA inhibited the expression of LC3-II/LC3-I, but had no effect on the expression of caspase-3, PARP or BCL-2.</p><p><b>CONCLUSION</b>Rapamycin can inhibit the proliferation, induce apoptosis and autophagy of RPMI 8226, the hydroxychloroquine can inhibit autophagy and proliferation of RPMI 8226, and induce apoptosis, the 3-MA can inhibit autophagy of RPMI 8226, but hardly has any effects on proliferation and apoptosis of RPMI 8226 cells.</p>

Significance of H3K27me3 and EZH2 in Predicting the Therapeutic Efficacy of Diffuse Large B-Cell Lymphoma / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate the significance of H3K27me3 and its methyltransferase EZH2 in predicting the short-term and long-term outcome of newly diagnosed patients with diffuse large B-cell lymphoma (DLBCL).</p><p><b>METHODS</b>The paraffin wax speciments of 102 DLBCL patients in Fujian Medical University Cancer Hospital were collected. The expression of H3K27me3, EZH2 and BCL-2 protein were detected using tissue array made by tissue microarray(TMA) technique and immunohistochemistry method. The evaluation data after clinical treatment and follow-up results were collected and combined with expression levels of H3K27me3, EZH2 and BCL-2 detected by tissue array, then on the basis of these data, the survival of patients was analyzed by Kaplan-Meier method, the correlation of EZH2 with H3K27me3 and BCL-2 was analyzed by pearson correlation test, the correlation of above mentioned indicators with different therapeutic efficacy was analyzed by spearman correlation test. The relationship of H3K27me3 and EZH2 expression as well as co-expression of H3K27me3 and EZH2 with the therapeutic efficacy and prognosis of patients were compared.</p><p><b>RESULTS</b>A total of 61.8% patients showed EZH2 high expression which positively correlated with high expression of H3K27me3 and BCL-2. The complete remission (CR) and overall remission (OR) rates in H3K27me3 high expression and co-expression of H3K27me3 EZH2 groups were lower than those in low expression groups (P<0.001), moreover OS and PFS rates also were lower than those in low expression (P<0.001). In the RCHOP subgroup, the patients with EZH2 low expression showed significantly better CR, OR OS and PFS in comparison with those of patients with higher expression (P=0.003,P=0.019).</p><p><b>CONCLUSION</b>Part of DLBCL patients with H3K27me3 high expression or coexpression of both H3K27me3 and EZH2 exhibit a worse prognosis in comparison with those patients with H3K27me3 low expression or without coexpression. The patients with EZH2 low expression usually responde well to RCHOP regimen in the short-term or long-term survival.</p>

Effect of Complicated Hemophagocytic Syndrome on Clinical Prognosis of Patients with Non-Hodgkin's Lymphoma and Analysis of Its Affecting Factors / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate the effect of complicatal hemophagocytic syndrome on clinical prognosis of patients with non-Hodgkin's lymphoma (NHL) and analyze its factors affecting prognosis.</p><p><b>METHODS</b>Ninety cases of NHL were selected and divided into 2 groups: 61 cases of NHL without hemophagocytic syndrome as group A and 29 cases of NHL with hemophagocytic syndrame as group B. The survival analysis of Kaplan-Meter method and the Cox regression model were used for univariate and multivariate analyses of related factors.</p><p><b>RESULTS</b>The patients in group B were more likely to start with fever, moreover, the hemophagocytes could be found in bone marrow samples of 89.66% (26/29) patients; the levels of total bilirubin, triglycerides, serum ferritin, serum soluble CD25, DNA copies of epstein-barr virus (EBV) and lactate dehydrogenase level in the group B were significantly higher than those in the group A(P<0.05). And the patients in group B had worse physical state, later disease stage, worse disease status and lower overall prognosis as compared with patients in the group A. The complicased hemophagocytic syndrome, incomplete improvemant of deseases state after treatment and EBV infection were the independent risk factors for the poor prognosis of patients with NHL.</p><p><b>CONCLUSION</b>The complicated hemophagocytic syndrome can increase the severity of NHL, there fore significantly influences the clinical prognosis of patients, while the complicated hemophagocytic syndrome, poor therapatic efficacy for patients and EBV infection are independent risk factors affecting the prognosis of NHL patients.</p>

Anti-proliferation Effect of NS-398 in Combination with Bortezomib on Multiple Myeloma RPMI 8226 Cells In Vitro / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate if NS-398 could enhance the chemosensitivity of Bortezomib (BOR) on human multiple myeloma RPMI 8226 cells.</p><p><b>METHODS</b>After the treatment of NS-398 combined with BOR, MTT assay was used to detect the proliferation inhibition effect on human multiply myeloma RPMI 8226 cells in vitro, Flow cytometry was used to analyze their effect of apoptosis and cell cycle; the caspase-3 activity of different treatment group was detected by using ELISA and the activity of Cox-2 was measured by using Cox-2 activity assay kit.</p><p><b>RESULTS</b>The inhibitory rate of NS-398 combined with BOR was higher than that of NS-398 or BOR alone(Q>0.85). After treatment of NS-398 combined with BOR, the percentage of cells arrested in the G/Gphase and apoptotic rate were both higher than that of treatment with each drug alone(Q>1.15). The caspase-3 activity in cells treated with combined of NS-398 and BOR was significantly higher than that of treatment of each drug alone(Q>1.15).</p><p><b>CONCLUSION</b>NS-398 combined with BOR shows a synergistic effect on the growth inhibition of RPMI 8226 cells in vitro.</p>

Expression and Prognostic Significance of H3K27 Trimethylation Protein in DLBCL / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate the expression and prognostic effect of H3K27 trimethylation protein (H3K27me3) in diffuse large B-cell lymphoma(DLBCL).</p><p><b>METHODS</b>A total of 102 DLBCL patients from Fujian Provincial Cancer Hospital were enrolled in this study. No therapy had been given before specimen collection. Tissue microarray(TMA) technique and immunohistochemistry(IHC) method were used for H3K27me3 immunostaining. Clinicopathologic and suvival data were carefully collected. The association between tested markers, clinicopathologic characteristics and prognosis were evaluated. Survival rates were analyzed by the Kaplan-Meier method, and prognostic factor were analyzed by the Cox proportional hazards model, the relation of different expression levels with clinical feature and prognosis of patients was compared.</p><p><b>RESULTS</b>The quality of TMA was perfect and meet the standard of analysis. Among all DLBCL patients, 59.8% were characterized with high expression of H3K27me3, correlated with age, ECOG≥2, extranodular disease number≥2, elevation of LDH, medium-high risk IPI. Patients with high H3K27me3 expression manifested that the complete remission rate(CR) and overall remission rate (OR) were lower than those of patients with low expression, i.e., 20% vs 57.5% and 41.8% vs 90%, respectively (P<0.001). In addition, patients with high H3K27me3 expression showed shorter median survival time, i.e., 21.5 mon (P<0.0001). Multivariate analysis indicated that H3K27me3 was an independent risk factor for DLBCL patients (P=0.007).</p><p><b>CONCLUSION</b>TMA technique is valid for the construction of DLBCL tissue chips. Patients with high expression of H3K27me3 indicates little response to treatment, worse outcome and shorter overall survival. The detection of H3K27me3 expression possesses a certain clinical value for prediction of DLBCL outcome.</p>

Ad-NK4 Enhances the Chemosensitivity of Human Multiple Myeloma RPMI 8226 Cells to Bortezomib / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate if Ad-NK4 can enhance the chemosensitivity of human multiple myeloma RPMI 8226 cells to bortezomib(BOR).</p><p><b>METHODS</b>The cell-matrix adhesion test and PRMI 8226 cell-ECV 304 cell adhesion test were used to analyze the effect of Ad-NK4 on adhesion of RPMI 8226 cells; Western blot was used to detect the expression changes of adhesion and invasion-associated proteins MMP2, MMP3, MMP7 and VEGF; MTT assay was used to detect the effect of Ad-NK4 on proliferation of RPMI 8226 cells; the flow cytometry with PI staining was used to detect the effect of Ad-NK4 on cell apoptosis; the expression of cleaved caspase-3, BAX and BCL-2 was assayed by Western blot.</p><p><b>RESULTS</b>These 2 adhesion assays indicated that Ad-NK4 significantly inhibited the adhesion of human multiple myeloma RPMI 8226 cells. In addition, Erk and JAK/STAT pathway may be involved in the process. The expression level of MMP-2, MMP-3 and VEGF were decreased in Ad-NK4 group, compared with untreated or Ad-GFP group (P<0.05). However, the expression of MMP-7 protein in Ad-NK4 group was not significantly different from untreated or Ad-GFP group (P>0.05). The inhibitory rates of the proliferation in cells treatedly Ad-NK4 combined with BOR was significantly higher than that with BOR or Ad-NK4 alone. Similarly, Western blot indicated that the level of cleaved caspase-3 and BAX in cells treated with Ad-NK4 combined with BOR was significantly higher than BOR or Ad-NK4 alone, but BCL-2 protein expression was significantly lower. Meanwhile, the ratio of BAX/BCL-2 was increased.</p><p><b>CONCLUSION</b>Ad-NK4 can enhance the chemosensitivity of human multiple myeloma RPMI 8226 cells to BOR,which is associated with increasing of both BAX/BCL-2 ratio and Caspase-3 activation.</p>

Inhibitory effect of bone marrow mesenchymal stem cells on lymphoma cell proliferation / 中国实验血液学杂志

Growing evidences show that mesenchymal stem cells (MSC) home to tumorgenesis and inhibit tumor cells, however, their molecular mechanisms are unclear. The purpose of this study was to explore the effect of B7-H4 in the influence of the mouse MSC on the proliferation of lymphoma cell line EL-4. The expression of B7-H4 on the MSC cell line C3H10T1/2 (C3H10) was detected by using immunofluorescence. FAM-siRNA was synthesized and transfected into C3H10 cells by INTERFER in (TM) siRNA Transfection Reagent. The transfection efficiency was determined by fluorescent microscopy and flow cytometry. The mRNA expression of B7-H4 was detected by RT-PCR after transfection of siRNA-B7-H4 into C3H10 cell line. The EL-4 was co-cultured with C3H10 siRNA-NC or C3H10 siRNA-B7-H4 for 48 hours, then was compared with EL-4 cultured alone, after 48 hours the cells were harvested under the confocal microscopy and measured by means of CCK-8 Kit. The results showed that the siRNA transfection efficiency in C3H10 cells reached to 72.43%, B7-H4 expressed highly on C3H10, the B7-H4 mRNA expression was down-regulated by transfection with different concentrations of siRNA into C3H10 cells. The proliferation of EL-4 was inhibited by C3H10 cells, and the effects were weakened and even disappeared after down-regulation of B7-H4. It is concluded that C3H10 can inhibit the proliferation of EL-4 through the expression of B7-H4, and this study provides new targets for the clinical treatment of lymphoma.

Effect of DAPT on proliferation and apoptosis of human multiple myeloma cell line RPMI8226 / 中国实验血液学杂志

The aim of this study was to explore the effect of DAPT (N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycinet-butyl ester) on proliferation in vitro of human multiple myeloma cell line RPMI8226 and its underlying mechanism. The proliferation of RPMI8226 cells was detected by CCK-8 method; flow cytometry was employed to assay the cell apoptosis rate;the expressions of Notch1 and Hes1 proteins were detected by Western blot. The results indicated that the proliferation of human RPMI8226 cells significantly decreased after treatment with DAPT 0.5 - 5.0 µmol/L for 24 - 72 h (P < 0.05) in a concentration- and time-dependent manner. DAPT significantly induced apoptosis of RPMI8226 cells (P < 0.05). The expressions of Notch1 and Hes1 proteins were gradually downregulated with the increase of DAPT concentration. It is concluded that the DAPT can inhibit the proliferation of RPMI8226 cells, which may be related with the down-regulation of the protein expression of Notchl and Hes1.

Calcium ionophore induces the differentiation of chronic myeloid leukemia cells into dendritic cells / 中国实验血液学杂志

The aim of this study was to investigate the ability of calcium ionophore (CI ) to induce the differentiation of CML cells into dendritic cells (DC), to analyze the P210 expression in DCs and to evaluate the stimulatory effect of CML-DC on production of cytotoxic activity against CML cells via activating the autologous T cells. The mononuclear cells were isolated from bone marrow of CML patients whose WBC counts were more than 30x10(9)/L when samples were collected, then the lymphocytes and monocytes were discarded by pouring out supernatant twice at different culture time point. Slightly adherent cells were cultured in RPMI 1640 containing 10% FCS, with or without CI (375 ng/ml) and GM-CSF (200 ng/ml) at 37 degrees C, 5% CO2, fully humidified atmosphere for 96 hours. The cell morphology was observed under the inverted microscope and electron microscope; the expression of CD antigens was analyzed with flow cytometry; the P210 expression was measured with Western blot. LDH assay was used to evaluate the effect of cultured CML cells (CML-DC) generating cytotoxic T lymphocyte (CTL) activity against CML cells. The results indicated that after treatment with calcium ionophore and GM-CSF for 96 hours, CML cells showed DC morphological characteristics under inverted microscope and electron microscope. The expression of CD83, CD86, CD40, CD80 and HLA-DR increased remarkably. P210 was expressed in the CML-DC, but the expression level was lower than that in CML cells without CI and GM-CSF treatment. LDH assay showed that the CTL activity against CML was found greater in autologous T cells activated by CML-DC than that by CML cells. It is concluded that the CML cells can be induced to quickly differentiate into DC when cultured with CI and GM-CSF. CML-DC expresses P210, but the expression level is lower than that in CML cells. CML-DC can stimulate autologous T cells to produce CTL against CML.

Relationship between heat shock protein 70-hom gene polymorphism and ankylosing spondylitis / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To investigate the association between heat shock protein 70-hom (HSP70-hom) gene polymorphism and ankylosing spondylitis(AS) in Chinese Han patients.</p><p><b>METHODS</b>Genomic DNA from 98 Chinese AS patients and 70 ethnically matched controls were typed for HSP70-hom polymorphism by polymerase chain reaction-restriction fragment length polymorphism.</p><p><b>RESULTS</b>The HSP70-hom genotypes in the AS patients consisted of homozygote AA (60.2%) and BB(4.1%), and heterozygote(35.7%), while the HSP70-hom genotypes in the controls were composed of AA(58.6%), BB(2.9%) and heterozygote(38.6%). No significant difference was found in the distribution of HSP70-hom genotype between these two groups(chi(2) test=0.280, P>0.05). The frequencies of HSP70-hom alleles in AS patients were 77.9%(AA) and 22.1%(BB), while they were 78.1% and 21.9% in the controls. The frequency of HSP70-hom allele in AS patients was not significantly increased, compared with that in controls (chi(2) test=0.002, P>0.05).</p><p><b>CONCLUSION</b>There may be no association between the HSP70-hom gene polymorphism and ankylosing spondylitis in Chinese Han population.</p>

Inhibition of K562 cell proliferation by wild type p16 and p53 genes co-transfection / 中国实验血液学杂志

The tumor suppressor gene p53 and p16, both of which play an important role in inhibition of tumorigenesis, are homozygously deleted in human myeloid leukemia cell line K562. To explore the inhibition of K562 cell proliferation by wild type p16 and p53 genes, both p16 and p53 genes were co-transfected into K562 cells mediated by liposome. The expression of the two genes was measured by immunocytochemical method, the cell cycle was analysed by flow cytometry, and the number of recovered viable cells was assessed after transfection. After co-transfection, the p53 and p16 positive cells were 23% and 28%, respectively. The results showed that co-transfection of p16 and p53 genes significantly inhibits cell proliferation comparing with transfection either by p16 gene or by p53 gene (P < 0.05). Expression of p16 and p53 proteins increased the cell number in G(1) phase but decreased the cell number in S phase. It is concluded that co-transfection of p16 and p53 genes has a stronger growth-inhibitory effect on K562 cell growth than that of transfection only by p16 gene or by p53 gene, may be a pathway for gene therapy in leukemia.

Bone Marrow Cells Activated by Autologous Dendritic Cells Purges Bone Marrow from Patients with Chronic Myelogenous Leukemia / 中国实验血液学杂志

In order to investigate the effect of autologous dendritic cells (DCs) activating bone marrow cells and purging bone marrow from chronic myelogenous leukemia (CML) patients, DCs were separated by negative selection system of human cells from bone marrow mononuclear cells (BMMNCs) of 2 CML patients in hematological remission and harvested after 3 days of culture in IMDM containing autologous plasma, rhGM-CSF and rhTNFalpha at 37 degrees C, 5% CO(2) humidified atmosphere. BMMNCs from the patients were also used to set up long-term culture (LTC) system in T-25 plastic flasks. The LTCs included three groups, i.e., control, addition of rhIL-2, and co-culture with autologous DCs. Half of non-adherent cells were collected, counted and assayed for CFU-GM weekly. Then, equivalent volume of fresh medium was replaced to maintain the culture. The culture was discontinued if the non-adherent cells count was less than 2 x 10(5). Adherent cells were collected for CFU-GM assay and flow cytometry for CD34 and P210. The colonies originating from the adherent cells were picked up under the inverted microscope. RNA was extracted, and BCR/ABL measured by nested reverse transcription polymerase chain reaction (RT-PCR). The results showed that the CFU-GM yields of non-adherent cells declined after 1 to 2 weeks co-cultured with autologous DCs, and it paralleled with group with rhIL-2. P210(+) cell percentage was also decreased. From the third week on, however, the decrease of CFU-GM yields slowed down, while CFU-GM in the system with rhIL-2 continued to fall. In system co-cultured with autologous DCs, the adherent cells contained the least percentagcs of CD34(+) cells and P210(+) cells percentage. However, the expression of BCR/ABL in CFU-GM colonies derieved from the adherent cells of DCs co-cultured had no significant difference with those from the culture without DCs. Our results suggest that co-culture of marrow cells with autologous DCs could significantly diminish the leukemic progenitors cells including both mature and primitive progenitor cells. Autologous dendritic cells might be used for ex vivo purging of CML marrow.