The study was aimed to investigate the expression of stromal cell derived factor (SDF-1) in bone marrow (BM) and its relation with apoptosis of BM CD34(+) cell and angiogenesis in myelodysplastic syndrome (MDS). 40 patients with MDS were divided into low-risk group and high-risk group according to IPSS score system. BM samples were collected. SDF-1 levels, the apoptosis of CD34(+) cells and microvessel density (MVD) of BM were detected by ELISA, flow cytometry and immunochemistry, respectively. The results showed that the SDF-1 level in MDS patients was significantly higher than that in normal controls(p < 0.05), and SDF-1 level in low-risk group was significantly higher than that in high-risk group. Apoptosis of CD34(+) cells significantly increased in low-risk group compared with other groups (p < 0.05). MVD in BM biopsy significantly increased in high-risk MDS group (p < 0.05), compared with low-risk MDS group which also had higher MVD than the control group (p < 0.05). Positive correlation was found between apoptosis of CD34(+) cells and SDF-1 levels in low-risk group, and SDF-1 level and MVD in high-risk group. It is concluded that the expression of SDF-1, apoptosis of BM CD34(+) cells and MVD were significantly abnormal in MDS patients, especially in different risk group, suggesting that SDF-1 level is related to cell apoptosis and angiogenesis.
Adolescent ; Adult ; Aged ; Apoptosis ; Bone Marrow ; metabolism ; Chemokine CXCL12 ; metabolism ; Child ; Female ; Humans ; Male ; Middle Aged ; Myelodysplastic Syndromes ; metabolism ; pathology ; Neovascularization, Pathologic ; Young Adult

The purpose of this study was to evaluate the biological behavior of stromal cell-derived factor-1 (SDF-1) in migration, adhesion and apoptosis as well as the related signaling transduction pathways in patients with myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). 37 patients with MDS, 10 patients with de novo AML and 14 patients with non-clonal cytopenia diseases were chosen for this study. The expression level of CXCR4 on CD34(+) cells and apoptosis of CD34(+) cells in bone marrow were detected by flow cytometry; the chemotaxis of SDF-1 on bone marrow mononuclear cells in 4 patients with low risk MDS (IPSS score ≤ 1.0) and 5 patients with high risk MDS (IPSS score ≥ 1.5) was assayed by transwell migration test of cells. The effect of SDF-1 on cell adhesion capability was measured by using CCK-8 method. The results indicated that the apoptosis rate of CD34(+) cells was significantly higher in MDS patients with low risk (IPPS score < 1.0) than that in MDS patients with high risk (IPSS score ≥ 1.5) (21.55% vs 7.52%, p < 0.001); as well, the apoptosis rate of CD34(+) cells was significantly higher in MDS patients with low risk than that in de novo AML patients (21.55% vs 7.33%, p < 0.001), no relation of CD34(+) cell apoptosis with age and sex of patients was found. SDF-1 could promote the cells of patients with CXCR4 high expression to adhere to the stroma cells, and induce migration of these cells, as well as, SDF-1 could trigger the polarization of the cells which highly expressed CXCR4. After addition of pertussis toxin, wortmannin and AMD3100, the ability of adhersion and migration of the cells with highly expressed CXCR4 decreased, but there was no above-mentioned phenomenon in patients who lowly expressed CXCR4. It is concluded that the SDF-1/CXCR4 axis enhances the ability of cell adhesion and migration through PI3K signaling pathway, thereby plays antiapoptosis role, moreover the above-mentioned effects can be blocked by PI3K pathway inhibitor and G protein inhibitor.
Apoptosis ; Bone Marrow Cells ; metabolism ; Cell Adhesion ; Cell Line ; Chemokine CXCL12 ; metabolism ; Humans ; Myelodysplastic Syndromes ; metabolism ; Receptors, CXCR4 ; metabolism ; Signal Transduction

In order to study the activity and gene expression of DNA methyltransferase (DNMT) on U266 myeloma cells and to analyze their significance, the activity of DNMT was detected by ELISA, and the expressions of DNMT1, DNMT3a and 3b were analyzed by RT-PCR. U266 cells were treated by phenylhexyl isothiocyanate (PHI), and the change of activity and gene expression of DNMT were determined. The results indicated that the activity and expression of DNMT in U266 myeloma cells were higher, compared with normal control. After being treated by different concentration of PHI, U266 cells were driven into apoptosis and the activity of DNMT decreased obviously and the mRNA level of DNMT declined. It is concluded that the activity and gene expression of DNMT on U266 myeloma cells are higher, and DNMT may be a new therapeutic target of multiple myeloma.
Cell Line, Tumor ; DNA (Cytosine-5-)-Methyltransferase 1 ; DNA (Cytosine-5-)-Methyltransferases ; metabolism ; DNA Methylation ; Humans ; Multiple Myeloma ; metabolism ; RNA, Messenger ; genetics

This study was aimed to explore the synergistic effect of 2-methoxyestradiol (2-ME2) and bortezomib (Bor) on the proliferative inhibition and apoptosis of U266 cell line and its possible mechanism. The cells were treated with 2-ME2, Bor alone and 2-ME2 combined with Bor, respectively. The cell viability and proliferative curve were detected by CCK8, the cell apoptosis was detected by caspase 3/7 activity test, cell cycle status was analyzed by flow cytometry, and real-time PCR was used to detect the mRNA expression of P21, BAX and BCL-2. The results showed that compared with cells treated with 2-ME2 or Bor alone, the proliferative potential of cells in combination group was significantly inhibited (p < 0.05), and apoptosis rate markedly increased (p < 0.05), cell cycle was arrested at G(1)-S phase, the mRNA expressive level of P21 and BAX increased, while the expression of BCL-2 decreased. It is concluded that 2-ME2 combined with Bor synergistically inhibits cell proliferation and induces apoptosis in U266 cell line. The possible mechanism may be associated with its effect of up-regulating P21 and BAX expressions.
Apoptosis ; drug effects ; Boronic Acids ; pharmacology ; Bortezomib ; Cell Line, Tumor ; drug effects ; Cell Proliferation ; drug effects ; Drug Synergism ; Estradiol ; analogs & derivatives ; pharmacology ; Humans ; Pyrazines ; pharmacology

The study was purposed to detect BAFF/APRIL gene expression changes in bone marrow mononuclear cells (BMMNC) and myeloma cell line U266 after interference with glucocorticoid and bortezomib. After separation of BMMNC from 7 patients with multiple myeloma, BAFF/APRIL mRNA expression in BMMNC and U266 cell line was detected by real-time PCR after treated with dexamethasone 100, 200 µg/ml, methylprednisolone 100, 200 µg/ml, bortezomib 0.1 µg/ml alone and dexamethasone or methylprednisolone combined with bortezomib respectively for 48 hours. The results showed that U266 cells and BMMNC of untreated MM patients highly expressed BAFF/APRIL genes. When dexamethasone, methylprednisolone or bortezomib was added to U266 cells or BMMNC alone, BAFF/APRIL gene expression decreased as compared with the blank control (p < 0.01). The inhibiting effect of bortezomib to BAFF/APRIL expression was obviously strong(p < 0.05). When dexamethasone or methylprednisolone combined with bortezomib, the BAFF/APRIL gene expression further decreased compared with dexamethasone or methylprednisolone alone (p < 0.01). As compared with the group of methylprednisolone combined with bortezomib, BAFF/APRIL gene expression decreased in dexamethasone combined with bortezomib with a statistically significant difference (p < 0.05). It is concluded that the expression of BAFF/APRIL gene is down-regulated after bing treated with glucocorticoids and bortezomib, which suggests that besides the glucocorticoid receptor and proteasomes targets, BAFF/APRIL and their receptor sites may be new targets of glucocorticoids and bortezomib.
B-Cell Activating Factor ; genetics ; metabolism ; Boronic Acids ; pharmacology ; Bortezomib ; Cell Line, Tumor ; Gene Expression Regulation, Neoplastic ; drug effects ; Glucocorticoids ; pharmacology ; Humans ; Multiple Myeloma ; metabolism ; Pyrazines ; pharmacology ; RNA, Messenger ; genetics ; Tumor Necrosis Factor Ligand Superfamily Member 13 ; genetics ; metabolism

The purpose of this study was to compare the efficacy of CEP plus G-CSF and CVP plus G-CSF regimens in the mobilization and collection of peripheral blood hematopoietic stem cells (PBHSC), and in the hematopoietic recovery. 57 patients with non-Hodgkin's lymphoma (NHL) underwent autologous PBHSC transplantation were analyzed retrospectively. The PBHSC were mobilized and collected by using CEP plus G-CSF and CVP plus G-CSF respectively, and were retransfused into these NHL patients after preconditioning, then the mobilization efficacy, adverse reactions and hematopoietic recovery were analyzed. The results showed that the WBC count decreased to ≤ 1.0 × 10(9)/L, platelet amount dropped to ≤ 40 × 10(9)/L during peripheral blood stem cell mobilization of all patients, which indicated successful collection of PBHSC. The mean value of (4.38 ± 3.40) × 10(8)/kg mononuclear cells (MNC) containing (2.79 ± 2.53) × 10(6)/kg CD34(+) cells were collected in CEP plus G-CSF group, while the mean value of (3.31 ± 1.23) × 10(8)/kg MNC containing (2.02 ± 0.87) × 10(6)/kg CD34(+) cells were collected in CVP plus G-CSF group. The efficacy of mobilization in CEP plus G-CSF group was significantly higher than that in CVP plus G-CSF group (p < 0.05). After preconditioning, bone marrow was suppressed in all patients. The average time of WBC count recovery to ≥ 1.0 × 10(9)/L was 11.4 days in CEP plus G-CSF group and 12.3 days in CVP plus G-CSF group; the average time of platelet amount recovery to ≥ 50 × 10(9)/L was 18.6 days in CEP plus G-CSF group and 19.3 days in CVP plus G-CSF group. The statistical analysis showed no significant difference in the average time of hematopoietic recovery between 2 groups. It is concluded that autologous PBHSC transplantation shows significant effect for treatment of patients with NHL. Either modified CEP or CVP plus G-CSF regimen is safe and effective in PBHSC mobilization. The CEP plus G-CSF regimen is better than CVP plus G-CSF regimen.
Adolescent ; Adult ; Child ; Female ; Hematopoietic Stem Cell Mobilization ; methods ; Humans ; Lymphoma, Non-Hodgkin ; therapy ; Male ; Middle Aged ; Peripheral Blood Stem Cell Transplantation ; Retrospective Studies ; Transplantation, Autologous ; Young Adult

This study was purposed to explore the feasibility of BIOMED-2 protocols for detection of immunoglobin (IG) and T-cell receptor (TCR) gene clonal rearrangement in bone marrow of Non-Hodgkin's lymphoma(NHL) patients, and to evaluate its clinical value. Gene clonal rearrangment (IGH, IGK, IGL, TCRβ, TCRγ, TCRδ) was detected by using BIOMED-2 protocols in 73 bone marrow examples of NHL patients. The PCR results were compared with the cytomorphologic examination of bone marrow. The correlation between PCR detection results and clinical stage, pathological factors were also evaluated. The results showed that clonal IG or TCR gene rearrangements were found in 31 of 73 cases (42.5%), higher than the positive rate of cytological analysis (24.7%, 18/73, p < 0.05). IG/TCR clonality rates were 40.0% (22/55) for B-NHL and 50% (9/18) for T-NHL. IG/TCR clonality rates detected in patients with III/IV stage were higher than those with I/II stage (p < 0.05). It is concluded that BIOMED-2 protocols are effective methods for detection of abnormalities in bone marrow in patients with lymphoma, and are superior to cytomorphologic examination. The positive rate of PCR detection is correlated with Ann Arbor stage, but is not related with malignant degree, age, treatment status, B symptoms or the involvement of spleen.
Adolescent ; Adult ; Aged ; Aged, 80 and over ; Bone Marrow ; pathology ; Female ; Gene Rearrangement, T-Lymphocyte ; Humans ; Immunoglobulins ; genetics ; Lymphoma, Non-Hodgkin ; genetics ; pathology ; Male ; Middle Aged ; Neoplasm Staging ; Polymerase Chain Reaction ; methods ; Young Adult

The aim of this study was to establish a method for quantitative detection of immunoglobulin heavy chain (IgH) gene rearrangements and to explore its clinical application in monitoring minimal residual disease (MRD) of acute lymphoid leukemia (ALL). Clonal IgH gene rearrangements in 51 patients with ALL at diagnosis were identified by multiplex PCR assay. PCR products were sequenced and pairwise in IMGT data base. Allele-specific oligonucleotides primers were designed complementary to the junctional region. The conservative JH primers combined with TaqMan probes were used to monitor the level of MRD in ALL patients. The sensitivity and specificity were assessed as well. The results indicated that out of all the 51 ALL patients, IgH rearrangements were identified in 21 cases, and 15 patients out of them were quantified. The slope of the standard curves was -3.1 to -3.9 and the correlation coefficients of all standard curves were > 0.98. The sensitivity was between 10(-4) and 10(-5), only one patient's sensitivity was 10(-3). Most of the quantitative range was less than 10(-4). The background's nonspecific amplification was detectable at a low level and had a little influence on results. 7 patients whose MRD level below 10(-3) kept complete remission and another 8 patients whose MRD level above 10(-3) had a higher relapse rate. It is concluded that the analysis of IgH gene rearrangements with RQ-PCR is a highly sensitive, specific and reliable method for accurate evaluation of MRD in ALL. The data indicates a high correlation between the level of IgH rearrangements and the prognosis in ALL patients.
Alleles ; DNA Primers ; genetics ; Female ; Humans ; Immunoglobulin Heavy Chains ; genetics ; Male ; Neoplasm, Residual ; diagnosis ; genetics ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; diagnosis ; genetics

This study was aimed to explore the effects of Notch ligand DLL4 on the protein expression of the transcription factor YY1 and proto-oncogene c-Myc, as well as K562 cell proliferation. The experiment was divided into 3 groups: normal control, negative control (pBudCE4.1-transfected) and experimental (pBudCE4.1-DLL4-transfected) groups. At 48 hours after transfection, the expression level of DLL4, YY1 and c-Myc proteins in K562 cells of each group were detected by Western blot and indirect immunocytochemical method; the CCK-8 method was used to detect proliferation of K562 cells; at 48 hours after transfection, cell cycle distribution and apoptosis of K562 cells were detected by flow cytometry. The results showed that the protein expression of DLL4, YY1 and c-Myc in K562 cells of every group were found. The protein expression levels of DLL4, YY1 and c-Myc in the experimental group cells were significantly higher than that in control groups (p < 0.05). The cell number in G(0)/G(1) phase increased in the experimental group and was higher than that in the control groups (p < 0.001), and the number of apoptotic cells were also increased (p < 0.001). It is concluded that DLL4 gene was successfully transfected into K562 cells, which increased the protein expression levels of transcription factor YY1 and proto-oncogene c-Myc, leading to the cell proliferation slower in experiment group, inducing the cell cycle arrested in G(0)/G(1) phase and increasing apoptosis.
Cell Cycle ; Cell Proliferation ; Humans ; Intercellular Signaling Peptides and Proteins ; genetics ; K562 Cells ; Proto-Oncogene Proteins c-myc ; metabolism ; Transfection ; YY1 Transcription Factor ; metabolism

This study was aimed to construct model cell line of NPM1-RNAi in HL-60 cells and its resistant line (HL-60/ADR) so as to provide a experimental basis for investigating the potential role of NPM1 gene in leukemia drug resistance. The shRNA targeting to NPM1 was ligated into linear pGCSIL-GFP vector, and transformed into E.coli DH5α. Positive clone was identified by PCR and DNA sequencing. pHelper 1.0, pHelper 2.0 and pGCSIL-GFP-NPM1-shRNA were cotransformed into 293T cells by lentivirus vector system. NPM1-RNAi-LV was transfected into HL-60 and HL-60/ADR cell lines. The efficiency of NPM-RNAi-LV was detected by using real-time quantitative RT-PCR and Western blot. The results showed that the recombinant eukaryotic expression vector pGCSIL-GFP-NPM1-shRNA was constructed. pGCSIL-GFP-NPM1-shRNA was packed into NPM1-RNAi-LV by lentivirus vector system, and transfected into HL-60 and HL-60/ADR cell lines. At mRNA level, the efficiency of NPM1 mRNA knockdown was more than 90% (p < 0.05). At protein level, obvious down-regulation of NPM protein was noted, indicating that NPM1 gene in HL-60 and HL-60/ADR cell lines was knocked down after transfected with NPM1-RNAi-LV. The resistance of HL-60/ADR cell line to adriamycin decreased to a certain degree after NPM1 gene silencing. It is concluded that the model cell lines of NPM1-RNAi in HL-60 and HL-60/ADR are successfully constructed, which can be used for investigating the potential role of NPM1 gene in drug resistance of leukemia.
Cell Line, Tumor ; Doxorubicin ; pharmacology ; Drug Resistance, Neoplasm ; genetics ; Genetic Vectors ; HL-60 Cells ; Humans ; Lentivirus ; genetics ; Nuclear Proteins ; genetics ; RNA Interference