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Unlabelled: Objetive: To investigate the effects of PKF118-310 on cell cycle and proliferation of K562 cell lines and its mechanism., Methods: After treatment of PKF118-310 with different concentration, the proliferation inhibition on K562 cell lines was detected by MTT, the existance of β-catenin and TCF-4 in the cells was observed by immunohistochemistry. The change of the cell cycle was detected by flow cytometry. The expressions of caspase-3, β-catenin, TCF and BCL-9 were detected by Western blot., Results: PKF118-310 can inhibit the proliferation of K562 cell line by S phase blocking. The β-catenin and TCF in the cells were observed by immunohistochemistry. After treating this cell line with PKF118-310 of different concentrations for 72 h, the expression level of caspase-3 increased, the expression levels of β-catenin, TCF and BCL-9 significantly decreased., Conclusion: PKF118-310 induces cycle arest of K562 cells at the S phase and inhibits the proliferation of these cells through decreasing β-catenin/TCF/BCL-9 thrascriptional activity.

To explore the relationship between expression of Foxp3 gene and immune activity of CD4(+) T cells, the Foxp3 gene was transfected with retroviral vector and applied to forcedly express Foxp3 protein in naive CD4(+)CD25(-) T cells, and then the effect of transfected CD4(+)CD25(-) T cells on immune co-stimulatory molecules and immune function of dendritic cells (DCs) was investigated, and the dependence of direct contact between Foxp3-transfected CD4(+)CD25(-) T cells and DCs was clarified by Transwell test. The results showed that through transfection of retroviral vector, CD4(+)CD25(-) T cells model expressing Foxp3 was established. At 1 week after transfection, proportion of T cells expressing Foxp3 was 38%. CD4(+)CD25(-) T cells forcedly expressing Foxp3 could play immune suppression role in vitro and induce down-regulation of CD80 and CD86 expression on the membrane of DCs. The lymphocyte proliferation test in vitro indicated that Foxp3 transfected CD4(+)CD25(-) T cells could inhibit effect of DCs on activation of allo-lymphocytes. It is concluded that the effect of Foxp3-transfected CD4(+)CD25(-) T cells on DC depends on intercellular direct contact.

The objective of this study was to investigate the change of native immune adhering function (ENIAF) in self-plasma of patients with hematologic and lymphoid neoplasms and its effect on the killing activity of NK cells. The whole blood was anticoagulated with citric acid. 5 microl precipitated red blood cells and 500 microl plasma of patients or controls were directly mixed with 750 microl quantitative K562 cells at 37 degrees C for 30 minutes. One K562 cell attached by one or more erythrocytes was counted as one rosette, the ratio of rosettes was calculated. Using K562 cells as target cells, the killing activity of NK cells isolated from normal persons was detected by MTT assay, the change of the killing activity was observed after adding RBCs. The results indicated that the ratio of rosettes formed by RBCs of 21 normal controls and K562 cells was 15.3% +/- 6.4%, and the ratio of rosettes formed by RBCs of 24 patients and K562 cells was 7.6% +/- 7.0%. The ability of ENIAF in patients with hematologic and lymphoid neoplasms was significantly lower than that in healthy individuals (t = 3.61, p < 0.001). The killing rate of NK cells in peripheral blood of normal individuals was 67% - 71% without adding RBCs, and it increased by 14.7% +/- 5.2% after adding RBCs of normal controls but decreased by 4.3% +/- 7.6% with RBCs of patients. It is concluded that the ENIAF of RBCs in patients with hematopoietic and lymphoid neoplasms decreases, accompanying the reduction of the killing activity of NK cells to K562 cells, so to detect change of ENIAF may be helpful for the assessment of the immunological function of patients with hematopoietic and lymphoid neoplasms.

In order to study the effects of phosphorothioated antisense oligodeoxynucleotides (ASODN) on the expression of VEGF in human lymphoma cell line Namalwa cells, human lymphoma cell line Namalwa cells were incubated with VEGF ASODN (the final concentrations of VEGF ASODN were 5, 10, 20 micromol/L respectively), or scrambled sequence for 24 or 48 hours. The expressions of VEGF mRNA and VEGF protein were detected by reverse transcriptase-polymerase chain reaction and streptavidin peroxidase (SP) immunohistochemistry respectively. The results showed that the expression levels of VEGF mRNA in Namalwa cells treated with three concentration levels (5, 10, 20 micromol/L of ASODN) were 1.38, 0.96 and 0.57 respectively. Those in PBS-treated cells and scrambled sequence treated cells were 1.79 and 1.84. When treated with 20 micromol/L VEGF ASODN for 48 hours, VEGF protein of Namalwa cells decreased greatly. Meanwhile, there was no obvious change in the scrambled sequence treated group. It is concluded that VEGF ASODN can suppress the VEGF expression in Namalwa cells in vitro.

In order to study how to induce tolerogenic dendritic cells in vitro and its mechanism, the K562 cells transduced with HLA-G construct were used to co-culture with DC. Then their related immunological changes, such as membrane molecules CD80, CD86, ILT3 and ILT4 expression levels were detected by flow cytometry. Allogeneic proliferation of peripheral blood mononuclear cells (PBMNC) was detected by mixed lymphocyte reaction. The results showed that CD80 and CD86 expressions on DC were downregulated, while ILT3 and ILT4 expressions were upregulated after co-culturing with K562-HLA-G cells. The DCs were less able to stimulate the allogenic PBMNC. It is concluded that the membrane-bound HLA-G can upregulate expression of inhibitory receptors ILT3 and ILT4, inducing tolerogenic DC in vitro, which may provide a novel strategy for transplant tolerance induction.

MRE11 plays an important role in the signal transduction of DNA damage response, therefore this study was purposed to explore the relationship between hMRE11 focus formation and DNA double-strand breaks (DSBs) caused by etoposide (VP-16) in human promonocytic cells U937. After U937 cells were treated with VP-16, the drug-induced DSBs were assessed by pulsed-field gel electrophoresis (PFGE), the gene transcription levels of hMRE11 were evaluated by RT-PCR, the nuclear focus formation of hMRE11 protein was examined using immunofluorescence technique, the cell cycle in parallel was analyzed by flow cytometry. The results showed that the percentage of U937 cells with DSBs induced by VP-16 raised from 13.0 +/- 2.3% in VP-16 2 microg/ml to 32.0 +/- 4.3% in VP-16 20 microg/ml (P < 0.01) along with increase of VP-16 dose. No difference of the hMRE11 mRNA level in U937 cells following the treatment with 100 microg/ml VP-16 at different times was discovered (P > 0.05). The hMRE11 protein was abundantly and uniformly distributed in the nuclei of untreated U937 cells outside of nucleoli, however, it formed discrete nuclear foci following VP-16 treatment. The mean value of nuclear foci increased by 5 to 20 times following the drug dosing (P < 0.01). An average of 5 nuclear foci per positive nucleus were observed at a dose of 2 microg/ml, and it was increased to an average of over 14 nuclear foci per positive nucleus after treating with VP-16 20 microg/ml. The percentage of nuclei containing hMRE11 nuclear foci also increased from less than 10% after treatment wiht VP-16 2 microg/ml to over 50% after VP-16 20 microg/ml (P < 0.01) following treatment with VP-16. After U937 cells were treated with 100 microg/ml VP-16 for 2 hours and fixed at 4, 8, 12 and 24 hours, the percentage of nuclei with hMRE11 nuclear foci increased to 61.54 +/- 3.6% (the control U937 cells: 0.47 +/- 1.17%, P < 0.01) at 8 hours, with a subsequent decrease in the percentage of nuclear foci-positive cells by 24 hours. The ratio of S-phase U937 cells at 8 hours after being treated with 100 microg/ml VP-16 for 2 hours was 47.55 +/- 2.35%, and that without 100 microg/ml VP-16 was 21.95 +/- 2.91% (P < 0.05). It is concluded that the nuclear focus formation of hMRE11 protein may be a response to DNA damage induced by topoisomerase II inhibitor VP-16 in human promonocytic cell line U937.

In order to investigate the inhibition role of anti-Fas hammerhead ribozyme on Fas expression and Fas-mediated apoptosis in CTLL-2 cells (mouse CTL cell line), and to explore a new way for enhancing the ability of T cells against Leukemia in donor lymphocytes infusion, CTLL-2 cells were transfected with pEGFP-RZ596 and pEGFPC1 (mock-transfected) via electroporation. Fas expression on CTLL-2 cells was detected by RT-PCR and Western blot. The killing effect of CTL against WEHI-3 (mouse acute myelomonocytic leukemia cell line) highly expressing FasL in vitro was detected by MTT assay. The caspase-3 proteolytic activity and the apoptosis rate of CTLL-2 cells were detected by means of BD AproAlert Caspase-3 Colorimetric kit and FITC labeled Annexin-V apoptosis detecting kit respectively. The results showed that the anti-Fas ribozyme could be successfully introduced into mouse CTLL-2 cells; Fas expression on the surface of cells transfected with the ribozyme was obviously decreased, in comparison with control and mock-transfected cells; after cocultured with WEHI-3 cells, the viability of CTLL-2 cells transfeced with the ribozyme was significantly increased, as compared with other two groups; caspase-3 activity and apoptosis rate of the ribozyme-transfeced cells were significantly decreased, the killing effect of CTLL-2 transfected with the ribozyme was stronger than that of other groups. It is concluded that anti-Fas ribozyme can remarkably decrease Fas expression on CTLL-2 cells, so as to avoid Fas-mediated apoptosis by Fas ligand on WEHI-3 cells, and to enhance their killing activity against WEHI-3 cells, as a result, the immune escape of acute myelomonocytic leukemia was depressed.

The purpose of this study was to investigate the expression of Fas, Fas ligand (FasL) and CD80 and function of FasL on the surface of acute myelomonocytic leukemia cells from WEHI-3 line. The expression of Fas, FasL and CD80 on the surface of WEHI-3 were detected by flow cytometry, the apoptosis of YAC-1 cell induced by FasL on the surface of WEHI-3 were detected by (3)H-TdR incorporation. The results showed that the expression rate of Fas, FasL and CD80 on the surface of WEHI-3 cells were (6.75 +/- 2.31)% (n = 5), (63.73 +/- 5.23)% (n = 5) and (5.06 +/- 0.41)% (n = 5) respectively. The apoptosis rate of YAC-1 cells (target cells) co-cultured with WEHI-3 cells (Effector cells) at the rate of 1:3, 1:10 and 1:30 were (26 +/- 4.5)%, (35 +/- 3.2)% and (43 +/- 2.7)% (n = 5) respectively. It is concluded that WEHI-3 cells have high expression of FasL and low expression of Fas and CD80 on their cell membrane, and can induce the apoptosis of Fas(+) YAC-1 cells.

This study was aimed to observe the biological characteristics of cryopreserved bone marrow mesenchymal stem cell (MSC) and to examine their abilities to support in vitro hematopoiesis. Bone marrow MSC were cryopreserved in -196 degrees C liquid nitrogen for 4 weeks (short term) and 9-15 months (medium term) with IMDM containing 10% DMSO, 40% fetal calf serum as cryoprotectant. The viability, proliferation, immunophenotype, in vitro differentiation and ability of supporting hematopoiesis of thawed MSC were investigated and compared with these of pre-cryopreserved MSC. The results showed that the cell viability were (93 +/- 2.51)% and (90 +/- 3.75)% for MSC cryopreserved as long as 4 weeks or 9-15 months respectively. However, there were no changes detected, as compared with pre-cryopreserved MSC in immunophenotype, abilities of proliferation and supporting colony forming of CFU-GM, CFU-E and CFU-GEMM. It is concluded that bone marrow-derived MSC can be stored in liquid nitrogen for short-term (4 weeks) or medium-term (9-15 months) without changes of abilities of proliferation, differentiation and hematopoiesis support.

To investigate the As(2)O(3)-chemosensitization of Gö6976 in K562 cells by its abrogation of As(2)O(3)-induced G(2)/M cell cycle arrest, K562 cells were treated with As(2)O(3) (5 micromol/L) and Gö6976 with various concentrations, the distributions of cell cycles were detected by flow cytometry, the cell viability was observed by trypan blue exclusion test and cell proliferation was tested by MTT assay. The results indicated that having treated by As(2)O(3) for 24 h and 48 h, the proportion of K562 cells in G(2)/M phase were (38.02 +/- 7.70)% and (32.58 +/- 7.43)% respectively, and no obvious cell apoptosis appeared. 50 nmol/L Gö6976 combined with As(2)O(3) decrease the proportion of cells in G(2)/M phase to (23.24 +/- 2.93)% and (16.18 +/- 1.60)% respectively and increase the proportion of cells in subG(1) phase to (11.82 +/- 2.31)% and (27.80 +/- 2.89)% respectively. Gö6976 abrogated G(2)/M cell cycle arrest induced by As(2)O(3) and increased cell apoptosis in a concentration- and time-dependent manner. Additionally, comparing to the control group, Gö6976 combined with As(2)O(3) decreased the cell viability and depressed the cell proliferation, but Gö6976 alone showed no same effect on them. In conclusion, the effects of AS(2)O(3)-chemosensitization of Gö6976 on K562 cells is associated with its abrogation of As(2)O(3)-induced G(2)/M cell cycle arrest.

Angiotensin II (Ang II) is one active substance of renin-angiotensin system. In order to explore the effect of Ang II combined with various cytokines on proliferation and differentiation of cord blood CD34(+) cells, in vitro experiments of cell cultures of Ang II with or without cytokines were taken place. The results showed that Ang II stimulated both BFU-E and CFU-GM expansion. The numbers of BFU-E and CFU-GM raised with increase of Ang II concentrations ranged from 0.01 - 0.1 micro mol/L. In semi-solid culture assay, Ang II stimulated CFU-GM production but no effect on BFU-E occurred. The multiple number of CFU-GM increased from 2.3 +/- 0.8 to 7.8 +/- 1.9 times when Ang II was added into SCF + G-CSF + GM-CSF + IL-3 combination. Similarly, the multiple number of BFU-E increased from 3.1 +/- 1.8 to 9.2 +/- 2.3 times when Ang II was combined with SCF + EPO + TPO + IL-3. In conclusion, Ang II stimulated cord blood hematopoietic stem/progenitor cell expansion in vitro the in presence of various cytokines.