Preliminary study on the effect of TRAIL on adhesion and apoptosis of multiple myeloma cell line RPMI8226 and its mechanism / 中国实验血液学杂志

The present study was purposed to investigate the inhibition effect of tumor necrosis factor-related apoptosis inducing ligand (TRAIL) on growth of RPMI8226 cells and adhesion between RPMI8226 cells and bone marrow stroma cells (BMSC), and to explore its mechanism as well. The inhibition effects of TRAIL on cells growth and adhesion were assayed by MTT; cell apoptosis was detected by Annexin V and PI; expression of genes bax, bcl-2, mcl-1, CARP1, CARP2, XIAP and cFLIP were determined by semi-quantitative RT-PCR; apoptosis-related protein expression was detected by Western blot. The results showed that TRAIL inhibited the proliferation of RPMI8226 cells in dose- and time-dependent manners. TRAIL induced apoptosis in RPMI8226 cells, the expression level of genes bcl-2, mcl-1, CARP1, CARP2, XIAP and cFLIP decreased, while the expression level of Bax increased, but the expression level of caspase-3 and NF-kappaB P65(RelA) proteins decreased. Moreover, TRAIL up-regulated the expression level of adherent molecule CXCR4 in RPMI8226 cells significantly. It is concluded that TRAIL up-regulated the expression level of adherent molecule CXCR4 in RPMI8226 cells significantly, and induced the apoptosis of RPMI8226 cells. Growth inhibition effect of TRAIL on RPMI8226 cells is in dose- and time-dependent manners.

Effect of a methylation inhibitor 5-aza-2'-deoxycytidine on SHP-1 gene expression, proliferation and apoptosis in K562 cells / 中国实验血液学杂志

The aim of this study was to investigate the regulation of 5-aza-CdR on transcription of SHP-1 gene and effects on the proliferation and apoptosis of K562 cells. Methylation-specific PCR (MSP) was used to detect CpG island methylation in SHP-1 promoter. MTT and flow cytometry were used to detect the growth and apoptosis of K562 cells after treatment with different concentration of 5-aza-CdR. The expressions of SHP-1 mRNA and protein were determined by FQ-PCR and Western blot. The expression of p-JAK2 was assayed by Western blot. The result showed that methylation of SHP-1 gene promoter was detected in K562 cells, and the SHP-1 mRNA and protein were expressed again in K562 cells after treatment with 5-aza-CdR, meanwhile the expression of phosphorylated P-JAK2 was down-regulated; 5-aza-CdR significantly inhibited the cell growth in dose and time dependent manners. AG490 inhibited the cell proliferation. 5-aza-CdR increased the apoptosis rate of K562 cells also in dose- and time-dependent manners. The apoptosis rates of K562 cells treated with 5-aza-CdR for 1, 3 and 5 days were 9.3%, 24.2% and 37.7% respectively. After treatment with 2 micromol/L 5-aza-CdR for 24 hours, cells in G(0)/G(1) phase increased gradually, cells in G(2)/M phase decreased gradually, cells were arrested in G(0)/G(1) phase. The cell ratios in G(2)/M phase at 1, 3 and 5 days after treatment with 5-aza-CdR were 30.7%, 23.45 and 19.3% respectively. It is concluded that the 5-aza-CdR, inhibitor of specific methylation transferase, can re-express the silent SHP-1 gene in K562 cells, inhibits the proliferation of leukemia cells and induces cell apoptosis by activating JAK/STAT pathway.

As2O3 induces demethylation and up-regulates transcription of SHP-1 gene in human lymphoma cell line T2 cells / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To investigate the methylation of CpG island in the SHP-1 gene promoter and its significance in lymphoma. To evaluate the effects of As2O3 on demethylation of SHP-1 in human lymphoma cell line T2 and on proliferation of T2 cells.</p><p><b>METHODS</b>T2 cells were treated with AsO3. Methylation specific PCR was used to detected the status of SHP-1 methylation in newly diagnosed lymphoma tissues and the T2 cells. The mRNA and protein expression of SHP-1 were determined by FQ-PCR and Western blot. The expression of phospha-c-kit was examined by Westren blot. MTT and flow cytometry were used to determine the growth and apoptosis in T2 cells.</p><p><b>RESULTS</b>T2 cells contained completely methylated SHP-1. Furthermore, there was constitutive c-kit phosphorylation. The expression of SHP-1 was recoverd when the cells exposed to AsO3, and concomitant with increasing SHP-1, a parallel down-regulation of phosphorylated c-kit occurred, so that by day 3 phosphorylated c-kit was barely detectable. As2O3 inhibited the cell growth, and the effects were dose- and time-dependent. As2O3 also increased apoptosis rate of T2 cells in a dose- and time-dependent manner, too, and on the 1, 2, 3 d treatment with AsO3 (2.5 micromol/L), the apoptosis rates were 6.12%, 26.53%, 50.90%, respectively. The frequency of methylation in SHP-1 gene promoter in lymphoma tissues was 87.5% (28/32). In the control group, however, 12 specimens of benign lymph node proliferation showed no methylation in CpG island of SHP-1 gene promoter.</p><p><b>CONCLUSION</b>Hypermethylation of SHP-1 gene promoter in lymphoma indicates the inactivation of SHP-1 gene and its possible role in the tumorigenesis of lymphoma. As2O3 can effectively cause demethylation and inhibit the growth of tumor by reactivating the SHP-1 gene transcription. SHP-1 methylation leading to epigenetic activation of c-kit may have a tentative role in the pathogenesis of lymphoma. Therefore, As2O3 is potentially useful in the treatment of lymphoma as a demethylating agent.</p>

Construction of expression plasmid for fused idiotypic DNA vaccine of B-cell lymphoma / 中国实验血液学杂志

The idiotypic determinant of surface immunoglobulin of B-cell lymphoma, as a tumor-specific antigen, has proved to be able to induce immune responses. To analyze whether an idiotypic vaccine fused with cytokine can elicit more effectively protective antitumor immunity, an eukaryotic expression plasmid was constructed, which encoded the fusion gene of single-chain variable fragment as a tumor specific antigen against B-cell lymphoma with monocyte chemotactic protein-3 (MCP3) as immunogen to elicit T-cell-dependent protective antitumor immunity, and EGFP (Enhanced Green Fluorescent Protein) gene as a marker to trace the survival, growth, differentiation and expression of the former exogenetic genes. The cDNAs for immunoglobulin (Ig) VH and IgVL were amplified by RT-PCR and assembled into the single-chain variable fragment (scFv) connected with a (Gly(4)Ser)(3) linker by recombinant PCR method. Then, the fragments of scFv and MCP3 were ligated with a NDAQAPKS spacer by the same method. The results showed that the fusion genes of scFv and MCP3-scFv were inserted into an eukaryotic expression vector pTARGET, and EGFP was cloned into the downstream of scFv and MCP3-scFv respectively. Finally the constructed plasmids were confirmed by sequencing and restriction analysis. In conclusion, a tumor-derived idiotypic DNA vaccine, encoding the fusion gene of single-chain variable fragment and monocyte chemotactic protein-3 (MCP3) to elicit a T-cell dependent, antitumor immunity, and the EGFP gene was inserted correctly. The DNA vaccine could be used for further study of DNA vaccine against B cell lymphoma in vivo.

Immunoglobulin D lambda multiple myeloma: a case report / 中国实验血液学杂志

To improve the recognition of immunoglobulin D multiple myeloma and explore its clinical feature and laboratory examination characteristics, so as to reduce the the missed diagnosis and misdiagnosis, a case of IgD multiple myeloma (MM) with myelofibrosis and bone marrow necrosis is reported. The clinical feature, treatment and prognosis of IgDlambda MM were discussed. Immunoglobulin D multiple myeloma is a rare disease and predominantly occurs in young male patients, which shows an aggressive clinical course with poor response to conventional treatment and unfavorable prognosis. Immunoglobulin D multiple myeloma was usually misdiagnosed as a light chain type multiple myeloma by using routine laboratory examination. Immunoglobulin D monoclonal protein is not easy to be detected owing to its low protein level, resulting in missed diagnosis. Immunofixation electrophoresis is highly sensitive and specific for diagnosis of IgD MM, can enhance accuracy of diagnosis for this rare disease.

Angioimmunoblastic T-cell lymphoma with autoimmune hemolytic anemia and pure red cell aplasia / 中国实验血液学杂志

Angioimmunoblastic T-cell lymphoma (AILT) is a peripheral T-cell lymphoma often complicated autoimmune phenomena such as autoimmune cytopenia, and is a truly rare type of NHL. In order to investigate the clinical features, pathological manifestation of this lymphoma, and to explore its therapy protocol, a 37-years old patient with AILT was investigated. The routine blood examination, bone marrow smear, lymphonodus biopsy, Coombs test, flow cytometry for bone marrow mononuclear cells, serological test, immunochemistry method etc were performed for this patient. The results showed that the systemic lymphadenectasis and hepatosplenomegaly were seen in patient, the cervical lymphonode biopsy revealed AITL. The hematoglobin level and number of reticulocytes were very low. Coombs test was positive. Simultaneously, the bone marrow aspirate revealed erythroid aplasia. The warm type autoimmune hemolytic anemia (AIHA) and pure red cell aplasia (PRCA) were co-existed. After one course of chemotherapy with CHOP-E, infiltration sign of AITL patient with AIHA and PRCA disappeared. In conclusion, the AITL patient complicated with AIHA and PRCA was successfully diagnosed, the lymphonode biopsy and bone marrow smear showed more significant, the chemotherapy protocol of CHOP-E can give some effect to cure such angioimmunoblastic T cell lymphoma.

Expression and mutation of myc antagonist genes Mad1, Mxi1 and Rox in leukemia cells / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the expression and mutation of Mad1, Mxi1 and Rox genes in leukemia cells.</p><p><b>METHODS</b>Expression and mutation of Mad1, Mxi1 and Rox genes in bone marrow mononuclear cells (BMMNC) from 26 de novo acute leukemia (AL) patients, and in peripheral blood mononuclear cells (PBMNC) from 30 healthy volunteers, as well as in 7 human leukemic cell lines were analyzed by reverse transcription-polymerase chain reaction (RT-PCR), single strand conformational polymorphism (SSCP) and DNA sequencing.</p><p><b>RESULTS</b>RT-PCR showed that all the above cells expressed Mad1, Mxi1 and Rox mRNA. SSCP revealed four polymorphisms: two in Mad1, one each in Mxi1 and Rox. DNA sequencing detected nine missense mutations: two in Mad1 in AL patients, four in Mxi1 (three in AL patients and one in KG-1 cell line), and three in Rox in AL patients. The mutations of Mad1, Mxi1 and Rox mRNA were detected in 2, 3 and 3 patients, respectively.</p><p><b>CONCLUSION</b>It is for the first time to demonstrate the mutations of Mad1, Mxi1 and Rox genes in AL patients suggesting these mutated genes involve in the pathogenesis of leukemia.</p>

Combined effect of recombinant mutant human TRAIL and daunorubicin in inducing apoptosis of leukemia cell and its mechanism / 中国实验血液学杂志

The aim of study was to investigate the combined effect of recombinant mutant human TRAIL (rmhTRAIL) with daunorubicin (DNR) or alone on K562 and U937 leukemia cell lines and its mechanism. The fibroblasts (MRC-5) of normal-human embryonic lung were used as control cells. After being treated with rmhTRAIL and DNR or only with rmTRAIL, the cytotoxic effect and the apoptosis rate in K562, U937 cells were measured by MTT assay. The expression levels of TRAIL death receptor and TRAIL decoy receptor mRNA in these three cell lines were assayed by semiquantitive RT-PCR before and after treatment with DNR. The results indicated that K562 and U937 were sensitive to rmhTRIAL. DNR had synergistic inhibitory effect with rmhTRAIL on the growth of K562 and U937 cell lines (P < 0.05). The expression level of DR4 and DR5 mRNA was significantly higher in K562 and U937 with combined treatment of rmhTRAIL and DNR than that in those alone, while the expressions of DcR1 and DcR2 mRNA were not influenced. It is concluded that in vitro, rmhTRAIL alone or in combination with DNR can obviously inhibit the growth of leukemia cell lines and induce cell apoptosis, DNR and rmhTRAIL have a synergistic inhibitory effect on growth of K562 and U937. The mechanism may correlate with the up-regulation of DR4 and DR5 of K562 and U937.

Construction and expression of a prokaryotic expression plasmid of idiotypic vaccine against B cell lymphoma: encoding the fusion genes of single-chain variable fragment and MCP-3 / 中国实验血液学杂志

The aim was to construct a prokaryotic expression plasmid encoding the fusion gene of single-chain variable fragment and monocyte chemotactic protein-3 (MCP-3). The cDNAs of immunoglobulin (Ig) VH and Ig VL were amplified by RT-PCR and assembled into the single-chain variable fragment (scFv) by recombinant PCR method. The cDNAs of Ig VH and Ig VL were connected by a (Gly4Ser)3 linker. Then, the fragments of scFv and MCP-3 were connected with a NDAQAPKS spacer, using recombinant PCR method again. The results indicated that the fusion gene of scFv-MCP-3 were constructed correctly and cloned into the prokaryotic expression plasmid successfully identified by sequencing and restriction endonucleases examination. Finally, the fusion protein was expressed in E coli DH5alpha under induction by arabinose. And the fusion protein was 65 kD and account for 30% of the total protein of the bacteria. In conclusion, a prokaryotic plasmid, encoding the fusion gene of single-chain variable fragment with MCP-3 and expressing idiotype protein vaccination against B cell lymphoma, was constructed correctly.

Relationship between VEGF and MMP-2, MMP-9 in 82 patients with acute myeloid leukemia / 中国实验血液学杂志

In order to investigate the relationship between VEGF and matrix metalloproteinase (MMP)-2, -9 in acute myeloid leukemia patients, and evaluate the significance of them in extramedullary leukemic invasion, the expressions of MMP-2 mRNA, MMP-9 mRNA, VEGF mRNA in bone marrow from 86 patients with acute myeloid leukemia (AML), as well as human hematopoietic cell lines were analyzed by reverse transcription-polymerase chain reaction (RT-PCR). The proteolytic activities of MMP-2 and MMP-9 in the supernatants were measured by zymography. The VEGF protein in serum of all samples was detected by ELISA. All these results were analyzed for determination of the relationship between VEGF and MMP-2, MMP-9. The results showed that there was a positive correlation between expressions of MMP-2 mRNA or MMP-9 mRNA and VEGF mRNA or protein. But no such correlation was demonstrated in the AML (CR) and normal control (NC) groups. A higher expression level of MMP-2 and MMP-9 in the VEGF positive group was found, as compared with the negative group (P < 0.05). More extramedullary infiltration occurred in VEGF positive groups than that in VEGF negative groups of AML. The expression of bcl-2 in HL-60 cells was upregulated by VEGF. It is concluded that there are significantly positive correlations between the expression of MMP-2 and MMP-9 with VEGF mRNA or protein levels in AML patients. VEGF can upregulate the expression of MMP-2, MMP-9 in HL-60 and a part of the primary leukemic cells. VEGF and MMP-2, MMP-9 may participate in the extramedullary leukemic invasion of AML patients.

Expression of midkine in patients with acute myeloid leukemia and its significance / 中国实验血液学杂志

The study was aimed to investigate the expression of midkine (MK) in bone marrow mononuclear cells (BM MNC) from 65 acute myeloid leukemia patients and 15 normal controls. The method of RT-PCR was used to examine the expression of MK mRNA in BM MNC. Parts of samples were incubated for 24 hours and the gene expression of MK in the BM MNC was detected by means of Western blot. The results showed that the expression of MK of BM MNCs in 50 newly diagnosed AML patients (0.331 +/- 0.436) and 15 AML patients in relapse (0.374 +/- 0.463) were markedly higher than that in 15 CR cases (0.067 +/- 0.190), and 15 normal controls (0), respectively. The complete remission in MK positive patients (63.16%) was significantly lower than that in MK negative group (93.55%). The patients with positive MK expression had a higher relapse rate than those with negative MK expression. The positive rate of MK gene expression in drug-resistant patients and drug-sensitive patients were 57.69% and 25.64% respectively and there was positive correlation between the gene expressions of MK and bcl-2 (P < 0.01) (r = 0.0556, P < 0.001). It is concluded that MK can be secreted by AML cells and involved in drug-resistant, its positive expression may be associated with the poor prognosis in newly diagnosed AML patients. The inhibitory effect of MK on apoptosis of leukemic cells is induced by upregulating bcl-2 expression.

WT1 gene expression lowered by IL-12 In vitro in peripheral blood mononuclear cells from patients with leukemia or myelodysplastic syndromes / 中国实验血液学杂志

Previous studies demonstrated that interleukin-12 (IL-12) enhances the non-MHC-restricted cytotoxic activity of NK cells and facilitate specific allogeneic human cytotoxic T lymphocyte responses against fresh leukemia cells and cell lines. The Wilms' tumor gene, WT1 mRNA, has been used as a marker of minimal residual disease (MRD) for evaluating therapeutic efficacy of patients with leukemia or myelodysplastic syndrome (MDS). This study was aimed to investigate whether in vitro IL-12 can lower WT1 gene expression in peripheral blood monuclear cells (PBMNC) from patients with leukemia or MDS. PBMNC from these 30 patients and 5 healthy volunteers were cultured at 5 x 10(5) cells/ml alone with or without 100 units/ml of IL-12 for 3 days. WT1 mRNA was measured by competitive reverse transcription polymerase chain reaction (RT-PCR) since WT1 mRNA is considered as a marker of minimal residual disease (MRD) in leukemia and MDS. The results demonstrated that WT1 mRNA in PBMNC of 5 healthy volunteers was less than 10(3) copies/microg of total RNA. Following the 3-day IL-12 treatment, mean WT1 mRNA of PBMNC was reduced from 10(4.8) to 10(4.2) copies/microg of total RNA in 6 CML patients, from 10(5.4) to 10(4.8) copies/microg in 12 MDS patients and from 10(5.0) to 10(4.2) copies/microg in 5 AML patients in CR, but not reduced in 5 of 7 AML in non-CR. It is concluded that IL-12 significantly decrease the quantity of leukemia cells in PBMNC of most patients with MDS, CML and AML in CR. IL-12 may be of considerable benefit in the elimination of MRD in patients with hematological malignancies.

A case of hepatosplenic gammadelta T cell lymphoma / 中国实验血液学杂志

Hepatosplenic gammadelta T cell lymphoma represents rare, often aggressive type of malignant peripheral T-cell lymphoma, which is characterized by expressing T-cell-associated markers CD2, CD3 and gammadelta T-cell receptor, and nonactivated cytotoxic cell phenotype (TIA-1+, granzyme B-). The pathological findings of a liver biopsy specimen revealed the diffuse infiltration of lymphocytes in the sinusoids and the aspiration biopsy from spleen revealed the diffuse infiltration of lymphocytes in the red pulp, not shaped to the nodes, often resulted in the misdiagnosis. Recently, by analyzing the immunophenotype and TCR rearrangement from liver, spleen and bone marrow, a case of adult hepatosplenic gammadelta T cell lymphoma was diagnosed. In combination with references, It is belived that immunophenotype and TCR rearrangement are necessary means to diagnosis hepatosplenic gammadelta T cell lymphoma.

Comparative study of expressions of cytoplasmic CD79a and other B-lymphoid immunomarkers in acute leukemic cells / 中国实验血液学杂志

To evaluate the expression of cytoplasmic CD79a (CyCD79a) and other commonly used B-lymphoid immunomarkers including cytoplasmic CD22 (CyCD22), CD19, CD20 and CD10 in various acute leukemia cells and to define the most sensitive and specific markers in the diagnosis of precursor B-cell acute lymphoblastic leukemia (pB-ALL), the immunophenotypic data from 221 de novo adult and pediatric acute leukemia patients as studied using multi-parameter flow cytometry in addition to routine morphologic and enzyme cytochemical assay, were retrospectively analyzed. Cytogenetic and/or molecular biological data in all 45 cases of acute promyelocytic leukemia (APL) and 13 cases of acute leukemia suspected as AML with the fusion genes such as AML1/ETO and CBFbeta/MYH11 were investigated. The results showed that CyCD79a and CyCD22 were the most sensitive and specific markers respectively for pB-ALL. Expression of CyCD79a was seen in 100% of 58 cases of pB-ALL. At the same time, none (0%) of all 147 cases of acute myeloid leukemia (AML) and 15 cases of precursor T-cell acute leukemia (pT-ALL) was positive for CyCD22. The conclusion is made that united detection of CyCD79a and CyCD22 is the optimal immune combination for the diagnosis pB-ALL and the distinguishing pB-ALL with AML and pT-ALL.

In vitro effects of mevastatin on the proliferation and apoptosis in human multiple myeloma cell line U266 / 中国实验血液学杂志

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

Mutation analysis of SHIP gene in acute leukemia / 中国实验血液学杂志

The SH2 domain containing inositol 5'-phosphatase (SHIP) was initially described as a 145 kD protein phosphorylated on tyrosines upon growth factor and cytokine stimulation. SHIP is predominately expressed in hematopoietic cells, and is a crucial negative regulator in the development of hematopoietic cells. To evaluate the role of the SHIP gene in human leukemogenesis, expression and mutation of SHIP gene in bone marrow and/or peripheral blood from 32 patients with acute myeloid leukemia (AML), 9 patients with acute lymphoblastic leukemia (ALL), as well as human hematopoietic cell lines were analyzed by reverse transcription-polymerase chain reaction (RT-PCR), single strand conformational polymorphism (SSCP) and sequencing. The RT-PCR showed that all samples expressed SHIP gene. Mutations of SHIP gene were detected in 7 out of 32 AML patients (22%) and one out of 9 ALL patients (12%). Interestingly, two missense mutations that had been observed in one AML patient at diagnosis disappeared after complete remission (CR). In addition, Akt phosphorylation was prolonged and increased following IL-3 stimulation in this patient sample. In conclusion, data of this study demonstrate the mutation of the SHIP gene in acute leukemia for the first time and suggest a possible role of the mutation of this gene in the development of acute leukemia. SHIP serves as a tumor suppressor by negatively regulating the PI3K/Akt signaling pathway in hematopoietic cells.

mitochondrial ceramidase overexpression up-regulates Bcl-2 protein level in K562 cells, probably through its metabolite sphingosine-1-phosphate / 中国实验血液学杂志

Recently, a mitochondrial ceramidase has been identified and cloned, whose mitochondrial localization strongly suggests the existence of an unexpected mitochondrial pathway of ceramide metabolism that may play a key role in mitochondrial functions, especially in the regulation of apoptosis. To explore the biological effect of mitochondrial ceramidase on cells, pcDNA 3.1/His-CDase plasmid, containing mitochondrial ceramidase cDNA sequence, was transducted into K562 cells mediated by liposome, and G418 was used to screen for positive colonies. A stable transfected K562 cell line was established and named as 'K562TC'. The difference between K562 and K562TC cells in chemotheraputic cytotoxicity response and serum-withdrawal resistance and Bcl-2 protein expression were evaluated by MTT assay, annexin V/PI test, flow cytometry or Western blotting, respectively. The results showed that although survival was comparable between K562 and K562TC cells after exposed to adriamycin, etoposide or arsenious acid, K562TC cells with elevated Bcl-2 protein expression level as identified by FCM or Western blotting revealed stronger resistance to apoptosis induced by serum withdrawal than their parental cells. Inhibition of mitochondrial ceramidase expression in K562TC cells by its specific antisense oligodeoxynucleotide was correlated with a decrease in Bcl-2 protein level. N, N-dimethylsphingosine, a sphingosine kinase inhibitor, depleted intracellular sphingosine-1-phosphate production, also abrogated Bcl-2 protein expression in K562TC cells, while Bcl-2 protein level in K562 cells was up-regulated by exogenous sphingosine-1-phosphate. It is concluded that mitochondrial ceramidase overexpression in K562 cells leads to markedly elevated level of Bcl-2 protein and results in more resistance to serum withdrawal. This effect is initiated not by sphingosine, the direct metabolite of mitochondrial ceramidase, but via sphingosine-1-phosphate, its phosphorylated form. This is the first evidence that mitochondrial ceramidase, through its sphingoid metabolite sphingosine-1-phosphate, up-regulates Bcl-2 protein expression in K562 cells.

Effect of panax notoginseng saponin on procoagulant activity and differentiation induction in NB4 cells / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To investigate the effect of Panax notoginseng saponin (PNS) on procoagulant activity (PCA) and differentiation induction in NB4 cells.</p><p><b>METHODS</b>After NB4 cells were treated with PNS, the recalcification time, PCA and TF-mRNA expression in NB4 cells were tested by RT-PCR. The inhibitory effect of PNS on NB4 cell proliferation was analysed by MTT method, NBT assay, cell morphological observation and flow cytometry.</p><p><b>RESULTS</b>(1) PNS of all concentrations could significantly prolong the recalcification time and lower the PCA level in NB4 cells in time-concentration-dependent manner. Simultaneously it down-regulated the expression of TF-mRNA. (2) PNS could partially inhibit the NB4 cell proliferation. (3) PNS could raise the NBT reducing capability of NB4 cells (P < 0.05). And morphological examination showed the differentiating tendency of monocyte and macrophage.</p><p><b>CONCLUSION</b>PNS could reduce the procoagulant activity and TF-mRNA expression in NB4 cells, and partially induce the differentiation of NB4 cells, therefore, it is hopeful to be a new anti-coagulant agent.</p>

Mutation analysis of SHIP gene in acute leukemia / 中华血液学杂志

<p><b>OBJECTIVE</b>The SH2 domain containing inositol 5'-phosphatase (SHIP) is predominately expressed in hematopoietic cells, and is a crucial negative regulator in the development of hematopoietic cells. This paper is to evaluate the role of the SHIP gene in human leukemogenesis.</p><p><b>METHODS</b>Expression of SHIP gene in bone marrow and/or peripheral blood from 32 patients with acute myeloid leukemia (AML), 9 with acute lymphoblastic leukemia (ALL), as well as human hematopoietic cell lines was analyzed by reverse transcription-polymerase chain reaction (RT-PCR), single strand conformational polymorphism (SSCP) and DNA sequencing.</p><p><b>RESULTS</b>RT-PCR showed that all samples expressed SHIP gene. Mutations of SHIP gene were detected in 7 (22%) of 32 AML patients and one (12%) of 9 ALL patients. Interestingly, two missense mutations that had been observed in a AML patient at diagnosis disappeared after complete remission (CR). In addition, in vitro Akt phosphorylation was prolonged and increased following IL-3 stimulation of this patient's cells.</p><p><b>CONCLUSION</b>Our data demonstrate for the first time the mutation of SHIP gene in acute leukemia and suggest a possible role of the mutation of this gene in the development of acute leukemia. SHIP may serve as a tumor suppressor by negatively regulating the PI3K/Akt signaling pathway in hematopoietic cells.</p>

Relationship between resistance to chemotherapy and expression of breast cancer resistance protein (BCRP) gene in patients with acute leukemia / 中国实验血液学杂志

In order to investigate the relationship between the expression of breast cancer resistance protein (BCRP) gene and drug resistance in patients with acute leukemia (AL), semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) was used to examine the expression of BCRP mRNA in AL patients and 15 normal subjects. Beta(2)-microglobin (beta(2)-MG) was used as positive reference. BCRP/beta(2)-MG ratio >or= 0.5 was defined as BCRP mRNA positive. The results showed that the positive percentage of BCRP gene expression in newly diagnosed group was 37.6%. The first complete remission rate was 79.3% and 31.6% in BCRP mRNA negative and BCRP mRNA positive patients respectively. The difference was significant (P = 0.001). The expression levels of BCRP mRNA in drug resistance group and drug sensitive group were 0.962 +/- 0.426 and 0.315 +/- 0.296 respectively (P = 0.0001). The expression level of BCRP mRNA in relapsed/refractory group was significantly higher than that in newly diagnosed group (P = 0.0025). The expression level of BCRP gene in normal individuals and long-term survival group was very low and correlated with FAB subtypes. It is concluded that high expression of BCRP mRNA leads to clinical drug resistance and is an unfavorable factor for prognosis of AL patients except acute promyelocytic leukemia.