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Effect of emodin on induction of apoptosis in jurkat cells and its possible mechanisms.

Tian-Nan WEI ; Jian-Da HU ; Ying-Yu CHEN ; Xin-Ji CHEN ; Ting-Bo LIU ; Lian-Huang LÜ

Journal of Experimental Hematology.2009;17(5):1203-1206.

The aim of study was to investigate the effect of a traditional Chinese medicine, emodin, on proliferation and apoptosis in T lymphocytic leukemic cell line Jurkat and its mechanisms. Cell proliferation inhibition was detected by MTT assay. Cell apoptosis was measured by DNA ladder and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay. The expressions of related proteins and caspase family members were determined by Western blot. The results showed that emodin inhibited proliferation in Jurkat cells, with an IC50 about 20 micromol/L and induced cell apoptosis in both time-and dose-dependent manners. The expressions of proliferation-related protein C-MYC, hTERT and apoptosis-related protein BCL-2 were down-regulated in a time dependent manner after the treatment with emodin. The expressions of procaspase-3, -8 and -9 all decreased while activated caspase-3 and PARP expressions were up-regulated. It is concluded that emodin can remarkably inhibit cell proliferation and induce apoptosis in Jurkat cells. The down-regulation of proliferation-related proteins C-MYC, hTERT and apoptosis-related protein BCL-2 expressions and activation of caspase cascade may be involved in the process of apoptosis.
Apoptosis ; drug effects ; Caspases ; metabolism ; Cell Proliferation ; drug effects ; Emodin ; metabolism ; pharmacology ; Humans ; Jurkat Cells

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Effects of baicalin on proliferation and apoptosis of adriamycin-resistant human leukemia HL-60/ADR cells.

Jing ZHENG ; Jian-Da HU ; Yi HUANG ; Bu-Yuan CHEN

Journal of Experimental Hematology.2009;17(5):1198-1202.

This study was purposed to explore the effects of baicalin on proliferation and apoptosis of adriamycin-resistant human myeloid leukemia cell line HL-60/ADR. HL-60/ADR cells were in vitro cultured and its proliferation inhibition was detected by MTT assay. The cell apoptosis was tested by Annexin V FITC/PI double staining analysis, DNA fragmentation and TUNEL labeling method. The expressions of c-myc and bcl-2 mRNA were detected by RT-PCR, and the protein expressions of C-MYC, BCL-2, caspase-3 precursor (procaspase-3), PARP and BAD were determined by Western blot. The results showed that baicalin could remarkably inhibited the HL-60/ADR cell proliferation, the cell doubling time was 48 hours, with an IC50 value of 28 micromol/L. Apoptosis occurred in dose dependent manner (20, 40, 80 micromol/L), and cell apoptosis in earlier and later stages could be detected by Annexin V FITC/PI double staining analysis, DNA fragmentation and TUNEL labeling method. The expressions of c-myc and bcl-2 mRNA in baicalin-treated cells decreased in a time-dependent manner (12, 24, 48 hours). Meanwhile, protein expressions of C-MYC, BBL-2, procaspase-3 and PARP (116 kD) were down-regulated in a time-dependent manner, while the expression of PARP (85 kD) and BAD were up-regulated. It is concluded that the baicalin efficiently induces proliferative inhibition and apoptosis in HL-60/ADR cells. All of above related genes and proteins may be involved in these processes.
Apoptosis ; drug effects ; Cell Proliferation ; drug effects ; Doxorubicin ; pharmacology ; Drug Resistance, Neoplasm ; drug effects ; Flavonoids ; pharmacology ; Gene Expression Regulation, Neoplastic ; HL-60 Cells ; Humans

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Calcium ionophore induces the differentiation of chronic myeloid leukemia cells into dendritic cells.

Hai-Rong ZHOU ; Jun-Min CHEN ; Zhi-Zhe CHEN ; Su-Wen WU ; Jiang-Rui GUO ; Yi-Qun HUANG

Journal of Experimental Hematology.2009;17(5):1192-1197.

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.
Antigens, CD ; metabolism ; Bone Marrow Cells ; cytology ; Calcium ; pharmacology ; Cell Differentiation ; Dendritic Cells ; cytology ; Female ; Granulocyte-Macrophage Colony-Stimulating Factor ; pharmacology ; Humans ; Ionophores ; pharmacology ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; pathology ; Male ; Monocytes ; cytology ; Tumor Cells, Cultured

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MDR reversal activity of bromotetrandrine in vitro and in vivo.

Jian CHENG ; Jue-Qiong WANG ; Bao-An CHEN ; Feng GAO ; Wen-Lin XU ; Hui-Lin SHEN ; Jia-Hua DING ; Chong GAO ; Yun-Yu SUN ; Jun WANG ; Gang ZHAO ; Hui-Hui SONG ; Wen BAO ; Qian SUN ; Yong-Yuan DAI ; Xin-Chen SUN ; Hong-Yan CHENG ; Yu-Xia DENG ; Guo-Hong LI ; Ning-Na CHEN ; Li-Jie LIU ; Xue-Mei WANG

Journal of Experimental Hematology.2009;17(5):1183-1191.

The present study was aimed to evaluate the MDR reversal activity of bromotetrandrine (BrTet) in vitro and in vivo. The inhibitory effects of adriamycin (ADM) used alone or in combination with BrTet or Tet on the proliferation of K562 and K562/A02 cells were evaluated by MTT assay. The ADM accumulation and the protein levels of P-glycoprotein (P-gp) were detected by flow cytometry. The mRNA levels of P-gp were determined by RT-PCR. The in vivo effect of BrTet and Tet was investigated by using nude mice grafted with sensitive human leukemia cell line K562 and MDR cell line K562/A02. The results showed that BrTet at 0.25, 0.5 and 1 micromol/L reversed the resistance to ADM in MDR K562/A02 cells in a dose-dependent manner. Flow cytometry suggested that BrTet significantly increased the intracellular accumulation of ADM in K562/A02 cells in a dose-dependent manner. BrTet also inhibited the overexpression of P-gp in K562/A02 cells, and down-regulated mdr1 expression. In nude mice bearing K562 xenografts on the left flank and K562/A02 xenografts on the right flank, intraperitoneal injection of 10 mg/kg BrTet significantly enhanced the antitumor activity of ADM against K562/A02 xenografts with inhibitory rates of 26.1%, while ADM alone inhibited the growth of K562/A02 xenografts only by 5.8%. No enhancement effect by BrTet was seen in K562 xenografts. It is concluded that BrTet shows significant MDR reversal activity in vitro and in vivo. Its activity may be related to the inhibition of P-gp overexpression and the increase intracellular accumulation of anticancer drugs. BrTet may be a promising-MDR modulator for eventual assessment in the clinic.
ATP Binding Cassette Transporter, Sub-Family B ; ATP-Binding Cassette, Sub-Family B, Member 1 ; metabolism ; Animals ; Benzylisoquinolines ; pharmacology ; Drug Resistance, Multiple ; drug effects ; genetics ; Drug Resistance, Neoplasm ; drug effects ; genetics ; Female ; Humans ; K562 Cells ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Xenograft Model Antitumor Assays

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Effect of tetrandrine combined with daunorubicin on expressions of P21 and P-gp in K562/A02 cells.

Bao-An CHEN ; Jing LI ; Jian CHEN ; Feng GAO ; Ya-Nan WU ; Min-Sheng ZHU ; Jia-Hua DING ; Chong GAO ; Xin-Chen SUN ; Wen-Lin XU ; Xue-Mei WANG

Journal of Experimental Hematology.2009;17(5):1179-1182.

This study was aimed to investigate the reversal effect of Tetrandrine (TET) combined with daunorubicin (DNR) on multidrug resistance (MDR) of K562/A02 cells and its relation to P21, P-gp and their genes so as to provide the new theoretic evidence for clinical use of TET. The experiments were divided into 4 groups: control group (DNR alone), combined 1 group (DNA+0.5 mg/L TET), combined 2 group (DNR+1.0 mg/L TET) and combined 3 group (DNR+2.0 mg/L TET). The expressions of P21, P-gp and mdr-1 gene in K562/A02 cells of different groups were detected by Western blot, flow cytometry and semi-quantitative PCR respectively. The results showed that the expression of P21 was enhanced along with increasing of TET concentration, the expression of P-gp was reduced along with increasing of TET concentration and expression of mdr-1 gene was almost not observed in K562 cells, but the high expression of mdr-1 gene was seen in K562/A02 cells, furthermore, the expression of mdr-1 gene in K562/A02 cells increasingly was reduced along with increasing of TET concentration. It is concluded that the TET possesses the reversal effect on multiple drug resistance of K562/A02 cells with concentration dependence, the reversal effect of TET may be related to up-regulation of P21 expression and down-regulation of P-gp and mdr-1 gene expressions in K562/A02 cells.
ATP Binding Cassette Transporter, Sub-Family B ; ATP-Binding Cassette, Sub-Family B, Member 1 ; metabolism ; Benzylisoquinolines ; pharmacology ; Cell Line, Tumor ; Cyclin-Dependent Kinase Inhibitor p21 ; metabolism ; Daunorubicin ; pharmacology ; Drug Resistance, Multiple ; Drug Resistance, Neoplasm ; Humans ; K562 Cells

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Immunophenotypes, cytogenetics and clinical features of 192 patients with acute myeloid leukemia.

Hai-Xia TONG ; Hui-Han WANG ; Ji-Hong ZHANG ; Zhuo-Gang LIU ; Ying-Chun ZHENG ; Yun-Xiu WANG

Journal of Experimental Hematology.2009;17(5):1174-1178.

The objective of this study was to investigate the immunophenotypic subtype profiles of 192 patients with acute myeloid leukemia (AML) and its association to cytogenetics and clinical features. Immunophenotyping of 192 patients was performed by flow cytometry using a panel of monoclonal antibodies. The karyotypes in 125 out of 192 cases were analyzed by G-banding technology. The results showed that CD33, CD13, myeloperoxidase (MPO) and CD117 were the most commonly expressed antigens in AML. CD117 expressed in 84.6% of AML-M3 cases. A combination of intensive autofluorescence, both CD34- and HLA-DR-, and high expression of CD13, CD33 and MPO had significant value for AML-M3 diagnosis. CD14 expressed only in AML-M4 and AML-M5, and both intensive positivity of CD64 and CD15 with high expression of HLA-DR may suggest great possibility for diagnosis of AML-M5. Lymphoid marker expression was documented in 47.9% of the 192 AML cases. CD56 (26.0%) and CD7 (20.8%) were the most commonly expressed lymphoid markers in AML patients, followed by CD19 (9.9%) and CD2 (7.3%). Abnormal karyotypes were detected in 76 out of 125 cases (60.8%). Correlation test showed that t(8;21) was found only in 17 cases of AML-M2 and strongly associated with the individual or combinational expressions of CD15/CD19/CD56. And 28 cases of t(15;17) were found in AML-M3; 2 cases of inv(16) were found in AML-M4EO. Higher CD34 positivity was found in LymAg+ group (77.2%) than that in LymAg- group (48.0%). It is concluded that immunophenotype analysis is useful for AML diagnosis and classification, and the immunophenotype has close relevance to the abnormal cytogenetic changes and clinical features in AML. The results suggested that a new prognostic scoring system that integrated the morphology, cytogenetic abnormalities and immunophenotype parameters would benefit the diagnosis, classification, and estimation of prognosis in AML patients.
Adolescent ; Adult ; Aged ; Aged, 80 and over ; Child ; Child, Preschool ; Cytogenetics ; Female ; Humans ; Immunophenotyping ; Infant ; Leukemia, Monocytic, Acute ; genetics ; immunology ; Male ; Middle Aged ; Young Adult

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Expression of genes psma6 and slc25a4 in patients with acute monocytic leukemia.

Yin-Xia CHEN ; Wen-Ping WANG ; Peng-Yu ZHANG ; Wang-Gang ZHANG ; Jie LIU ; Xiao-Rong MA

Journal of Experimental Hematology.2009;17(5):1168-1173.

The aim of this study was to investigate the expression levels of genes psma6 and slc25a4 in bone marrow of patients with acute monocytic leukemia and their correlation with clinical features and prognosis. The expression levels of genes psma6 and slc25a4 in AML-M5 leukemia cells, normal blood cells and non-leukemia cells were detected by real-time quantitative RT-PCR and compared each other. The expression levels of psma6-encoding protein P27K was assayed by using immunohistochemistry method. The results showed that the expression levels of psma6 mRNA in AML-M5 leukemia cells was lower than that in non AML-M5 leukemia cells, non-leukemia cells and normal blood cells. The results obtained by immunohistochemistry assay were consistent with above-mentioned results. The expression level of psma6 in AML-M5 patients with complete remission was higher than that in AML-M5 patients without remission. The expression level of P27K protein in AML-M5 and AL correlated to leukocyte count in peripheral blood and LDH content. The overexpression of slc25a4 mRNA was found in AML-M5, but there was no significant difference in slc25a4 mRNA expression between the patients with complete remission and those without remission. It is concluded that the expression level of psma6 is probably a new prognostic indicator of acute monocytic leukemia, slc25a4 may be a novel gene of antigen associated with acute monocytic leukemia.
Adenine Nucleotide Translocator 1 ; genetics ; metabolism ; Adult ; Bone Marrow ; metabolism ; Female ; Humans ; Leukemia, Monocytic, Acute ; genetics ; metabolism ; Male ; Proteasome Endopeptidase Complex ; genetics ; metabolism

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Analysis of cells in Tel/aml-1 positive childhood acute lymphoblastic leukemia by two-dimensional gel electrophoresis.

Ye GUO ; Bao-Li FAN ; Yu-Mei CHEN ; Ying HU ; Yao ZOU ; Xiao-Juan CHEN ; Li ZHANG ; Xiao-Fan ZHU

Journal of Experimental Hematology.2009;17(5):1163-1167.

This study was aimed to establish the protein expression profile of tel/aml-1+ childhood acute lymphoblastic leukemia (ALL) with different prognoses by two-dimensional polyacrylamide gel electrophoresis and explore the nosogenesis of tel/aml-1+ ALL childhood. On the basis of leukocyte count at new diagnosis, early reaction to therapy and clinical prognosis, the patients with tel/aml-1+ ALL were divided into 3 groups: early relapse group, high leukocyte count group and standard risk group. The bone marrow was taken from newly diagnosed patients for isolating protein, then the protein in leukemia cells was isolated by two-dimensional polyacrylamide gel electrophoresis, the image analysis of differential protein among 3 groups was carried out by using the PDQuest 7.3.0 image analysis software. The results showed that there were significant difference of protein expression profile among 3 groups. As compared with high leukocyte count and standard risk groups, 71 protein spots disappeared, 93 new protein spots appeared, the expression of 37 protein spots was up-regulated and the expression of 23 protein spots was down-regulated in early relapse group. As compared with high leukocyte count group, 6 protein spots disappeared, 56 new protein spots appeared, the expression of 7 protein spots was up-regulated, and expression of 19 protein spots was down-regulated in standard risk group. It is concluded that the protein expression profile in early relapse group is significantly different from other groups. Some proteins may play an important role in pathogenesis of childhood tel/aml-1+ ALL, and probably become new molecular indicators and targets for individualized treatment.
Child ; Child, Preschool ; Core Binding Factor Alpha 2 Subunit ; metabolism ; Electrophoresis, Gel, Two-Dimensional ; Female ; Humans ; Male ; Oncogene Proteins, Fusion ; metabolism ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; metabolism ; pathology

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Abnormality of serum immunoglobulin in peripheral blood of patients with chronic lymphocytic leukemia.

Yuan-Dong ZHU ; Wei XU ; Kou-Rong MIAO ; Xin CAO ; Lei FAN ; Qiong LIU ; Lin YAO ; Yu-Jie WU ; Min HONG ; Jian-Yong LI

Journal of Experimental Hematology.2009;17(5):1159-1162.

In order to investigate the relation of serum immunoglobulin (Ig) level to age, sex, disease stages and prognosis in chronic lymphocytic leukemia (CLL) patients, the levels of serum IgG, IgA and IgM in 83 CLL patients were detected by immune rate turbidimetry. the expressions of CD38 and ZAP-70 in CLL cells were determined by multi-parameter flow cytometry (FCM). The results showed that among the 83 CLL patients, the IgG, IgA and IgM levels were reduced in 12 (14.5%), 26 (31.3%) and 34 cases (41.0%) respectively. The incidence of Ig reduction was higher in Binet C stage than that in Binet A and B (p=0.011). There was higher incidence of Ig reduction in high-risk group of Rai stage than that in low-risk group (p=0.011). The positive rate of CD38 or ZAP-70 was significantly higher in Ig reduction group than that in normal Ig level group (p=0.033 and p=0.038 respectively). The positive rate of CD38 and ZAP-70 was also higer in advanced disease, and among them the positive rate of ZAP-70 expression in Binet C stage was obviously higer than Binet A and B (p=0.047). Nonetheless, there was no significant relationship of Ig level with gender and age (p>0.05). It is concluded that the function of humoral immunity in CLL patient is closely related to the disease stages. The serum Ig detection seems important for evaluating immunologic state and prognosis of CLL patients.
ADP-ribosyl Cyclase 1 ; metabolism ; Adult ; Aged ; Aged, 80 and over ; Female ; Humans ; Immunity, Humoral ; Immunoglobulin A ; blood ; Immunoglobulin G ; blood ; Immunoglobulin M ; blood ; Immunoglobulins ; blood ; Leukemia, Lymphocytic, Chronic, B-Cell ; blood ; immunology ; Male ; Middle Aged ; Prognosis ; ZAP-70 Protein-Tyrosine Kinase ; metabolism

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Promotive effect of LRP16 gene on proliferation of K562 cells.

Bo YANG ; Xue-Chun LU ; Xiao-Hua CHI ; Wei-Dong HAN ; Li YU ; Fang-Ding LOU

Journal of Experimental Hematology.2009;17(5):1154-1158.

The study was aimed to investigate the promotive effect of LRP16 gene on K562 cell proliferation. Open reading frame of LRP16 gene was amplified using reverse transcription-polymerase chain reaction (RT-PCR) and ligated to pGEM-T plasmid to construct LRP16 ORF-pGEM-T recombinant vector. Then, LRP16 ORF identified by sequencing was inserted into pcDNA3.1+ plasmid to construct LRP16 ORF-pcDNA3.1+ recombinant expression plasmid which was transfected into K562 cell lines to make overexpression of LRP16 gene in K562 cells. Survival of cells was determined by MTT assay and growth curve of cells was drawn, the cell cycle was detected by flow cytometry. The results showed that LRP16 ORF was successfully amplified, then the LRP16 ORF-pcDNA3.1+ recombinant plasmid was constructed. The K562 cell line with overexpression of LRP16 gene was established. The promotive effect of LRP16 gene overexpression on proliferation of K562 cells was observed and the effect partially related to the enhancement of cells from G0 to S phase induced by LRP16 gene. It is concluded that LRP16 gene overexpression shows a promotive effect on proliferation of K562 cells.
Cell Proliferation ; Genetic Vectors ; Humans ; K562 Cells ; Neoplasm Proteins ; genetics ; Open Reading Frames ; Plasmids

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