[Effect of RITA on TP53 Mutant Human Mantle Cell Lymphoma Cell Line and Its Mechanism].

OBJECTIVE: To investigate the effect of RITA on TP53 mutant human mantle cell lymphoma (MCL) cell line Mino and its possible mechanism. METHODS: Mino cells were cultured in RPMI-1640 and treated with RITA at a concentration of 0-16 µmol/L for 24,48,72 hours. Cell viability was assessed by CCK-8 assay. The cells were treated by RITA (0-8 µmol/L) for 48 h, the cell apoptosis induced by RITA was detected by annexin V/PI flow cytometry. Western blot was performed to evaluate the expression of protein BCL-2, Caspase-3, Cleaved Caspase-3, PARP, MDM2, and P53 in Mino cells. RESULTS: After treatment with 0.5, 1, 2, 4, 8, and 16 µmol/L RITA for 48 h, the proliferation inhibition rate of Mino cells was (1.2±5.6)%, (14.9±4.9)%, (41.7±5.0)%, (61.8±2.4)%, (70.2±2.8)%, and (70.8±2.4)%, respectively. RITA could inhibit the proliferation of Mino cells significantly, and statistical analysis showed that the inhibition rate was increased with the increasing of RITA concentration (r=0.767). After the cells were treated by 4 µmol/L RITA for 24, 48, and 72 h, the proliferation inhibition rate was (25.2±3.8)%, (61.8±2.4)%, and (87.0±0.7)%, respectively. Satistical analysis showed that the inhibition rate was also increased with the increasing of treatment time (r=0.978). The apoptosis rate of Mino cells treated by 0, 2, 4, and 8 µmol/L RITA for 48 h was (5.4±0.4)%, (15.3±0.6)%, (38.7±1.7)%, and (50.8±1.1)%, respectively, and it showed dose-dependent manner (r=0.961). Western blot showed that with the increasing of RITA concentration, the BCL-2 protein expression was decreased in a dose-dependent manner (r=0.932), moreover, PARP cleavage and Caspase-3 activation were found, while the protein expression of MDM2 and P53 showed no change. CONCLUSION: RITA can inhibit the proliferation and induce the apoptosis of Mino cells significantly. The mechanism may be dependent on the Caspase pathway, but independent on the P53 pathway.

[Effect of bortezomib in inducing apoptosis of imatinib-resistant K562 cells and the mechanism].

OBJECTIVE: To investigate the effect of bortezomib in inducing apoptosis in imatinib-resistant K562 (K562R) cells and its possible mechanism. METHODS: K562 cells were cultured in gradient concentrations of imatinib for several months to generate imatinib-resistant K562 cells. The viability of K562R cells treated with bortezomib was measured using CCK-8 cell proliferation assay, and the cell apoptosis was analyzed by flow cytometry with annexin V/PI dual staining. Western blotting was used to detect the protein expressions of Mcl-1,Bcl-2 and Bcr/Abl. RESULTS: K562R cell line was successfully established, which showed 31.8 folds of imatinib resistance compared with the na?ve cells. Bortezomib treatment produced dose- and time-dependent inhibitory effect on the proliferation of both K562 cells and K562R cells and dose-dependently induced apoptosis in K562R cells. Combination of bortezomib with imatinib significantly enhanced the apoptosis of the cells. Western blotting showed that bortezomib treatment dose-dependently decreased the protein levels of both Mcl-1and Bcr/Abl in K562R cells without affecting bcl-2 protein expression. CONCLUSION: Bortezomib can inhibit the proliferation of K562R cells and induce cell apoptosis possibly by down-regulating Mcl-1 and Bcr/Abl expression and enhancing Mcl-1 cleavage.

[MiR-181a Promotes Proliferation of Human Acute Myeloid Leukemia Cells by Targeting ATM].

OBJECTIVE: To investigate miR-181a function and regulation mechanism by identifying miR-181a target genes in acute myeloid leukemia (AML). METHODS: The HL-60 cells of human AML was transfected by small molecular analog miR-181a, the cell proliferation was detected by CCK-8 method after electroporation in HL-60 cell lines. Target genes of miR-181a were predicted and analyzed by the bioinformatics software and database. Target genes were confirmed by HL-60 cell line and the patient leukemia cells. RESULTS: Overexpressed miR-181a in HL-60 cell line significantly enhanced cell proliferation compared with that in control (P < 0.05). Dual luciferase reporter gene assay showed that miR-181a significantly suppressed the reporter gene activity containing ATM 3'-UTR by about 56.8% (P < 0.05), but it didn't suppress the reporter gene activity containing 3'-UTR ATM mutation. Western blot showed that miR-181a significantly downregulated the expression of ATM in human leukemia cells. It is also found that miR-181a was significantly increased in AML, which showed a negative correlation with ATM expression. CONCLUSION: miR-181a promotes cell proliferation in AML by regulating the tumor suppressor ATM, thus it plays the role as oncogene in pathogenesis of AML.

[Correlation of donor and recipient serum interleukin 2 and tumor necrosis factor alpha levels to acute graft-versus-host disease in hematopoietic stem cell transplantation].

OBJECTIVE: To study the relationship between the donor and recipient serum interleukin 2 (IL-2) and tumor necrosis factor alpha (TNF-alpha) levels and the occurrence of acute graft-versus-host disease (aGVHD) following hematopoietic stem cell transplantation. METHODS: Twenty-two leukemic patients undergoing allo-hematopoietic stem cells transplantation and their donors were examined for serum levels of IL-2 and TNF-alpha using ELISA during conditioning and after the transplantation. IL-2 and TNF-alpha levels were also detected in the donors during mobilization to analyze the relationship between the cytokines and aGVHD. RESULTS: Five recipients showed no aGVHD, 10 developed grade I aGVHD, and 7 developed grade II-IV aGVHD. The serum levels of IL-2 and TNF-alpha after conditioning and post-transplantation were significantly higher in the recipients with grade II-IV aGVHD than in those with grade 0-I aGVHD, but no difference was found before the pre-conditioning. The serum IL-2 levels in the mobilized donors for the recipients with grade II-IV aGVHD were significantly higher than that in donors for recipients with grade 0-I aGVHD, whereas the levels of TNF-alpha showed no such a difference. CONCLUSION: Serum IL-2 and TNF-alpha levels in the leukemic patients after conditioning and post-transplantation, and the donor serum IL-2 level after mobilization may be correlative to the severity of aGVHD and can be used as indicators for predicting the severity of aGVHD after the transplantation.

[In vitro study of cytotoxic T lymphocyte activation by antigen-loaded dendritic cells for killing of K562 cells].

OBJECTIVE: To study the effect of dendritic cells (DC) stimulated with K562 cell lysate in inducing specific cytotoxic T lymphocytes (CTL) against K562 cells in vitro. METHODS: The DCs were derived from healthy human peripheral blood monocytes in the presence of granulocyte-macrophage colony-stimulating factor, interleukin (IL)-4 and tumor necrosis factor (TNF) alpha. The T cells were stimulated by DCs loaded with freeze-thawed K562 cells and T-cell cytotoxicities were measured by lactate dehydrogenase (LDH) assay. RESULTS: The DCs could be successfully obtained from peripheral blood monocyte after the culture. Mixed lymphocyte reactions induced by the antigen-loaded DC were much stronger than those induced by human peripheral blood monocytes (P<0.05). At the effector to target ratio of 10:1 and 20:1, cytotoxicities against K562 cells by CTL derived from culture with the antigen-loaded DCs were the strongest (P<0.05). CONCLUSION: CTL derived from DCs pulsed with K562 cell lysate show effective and specific cytotoxicity against K562 cells.