Effect of RITA on TP53 Mutant Human Mantle Cell Lymphoma Cell Line and Its Mechanism / 中国实验血液学杂志

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 / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the effect of bortezomib in inducing apoptosis in imatinib-resistant K562 (K562R) cells and its possible mechanism.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>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.</p>

MiR-181a Promotes Proliferation of Human Acute Myeloid Leukemia Cells by Targeting ATM / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate miR-181a function and regulation mechanism by identifying miR-181a target genes in acute myeloid leukemia (AML).</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>miR-181a promotes cell proliferation in AML by regulating the tumor suppressor ATM, thus it plays the role as oncogene in pathogenesis of AML.</p>

Recurrent deep vein thrombosis caused by heterozygous missense mutation of the protein S gene: genetic analysis of a case / 南方医科大学学报

<p><b>OBJECTIVE</b>To analyze the molecular pathogenesis of protein S deficiency in an adolescent case of recurrent deep vein thrombosis (DVT).</p><p><b>METHODS</b>Blood samples from the patient and his family members were collected for detection of the coagulation parameters by one-step clotting method, and the protein S (PS) and protein C activities were measured by a chromogenic assay. Enzyme-linked immunosorbent assay was employed for detecting the levels of free PS antigen. All the exons and exon-intron boundaries of the patients PS gene were amplified using PCR and analyzed by direct sequencing.</p><p><b>RESULTS</b>As carriers of hereditary PS deficiency, both the patient and his father showed a heterozygous C82792T point mutation in the 10th exon of their PS gene which resulted in the substitution of arginine314 by cysteine in the polypeptide chain of PS protein.</p><p><b>CONCLUSION</b>Recurrence of DVT in this patient is the result of hereditary PS deficiency caused by a novel heterozygous missense mutation in the PS gene.</p>

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 / 南方医科大学学报

<p><b>OBJECTIVE</b>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.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>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.</p>

Antithrombin deficiency due to heterozygous antithrombin gene mutation and a pedigree study / 中华血液学杂志

<p><b>OBJECTIVE</b>To identify the antithrombin (AT) phenotype and gene mutation of a kindred with hereditary antithrombin deficiency.</p><p><b>METHODS</b>Plasma AT activity and AT antigen level of the propositus and his kindred members were determined with chromogenic substrate method and immunoassay, respectively. All the seven exons and intron-exon boundaries of antithrombin gene were analyzed by PCR and direct sequencing of amplified PCR products from the propositus.</p><p><b>RESULTS</b>The propositus AT antigen level was normal but his AT activity was only 65% of normal value suggesting that he had type II AT deficiency. A heterozygous G13830A mutation in exon 6 resulting in Arg393His missense mutation in his AT polypeptide was identified in the propositus. The same phenotype and gene mutation were found in other 3 kindred members.</p><p><b>CONCLUSION</b>The type II AT deficiency found in this kindred is caused by heterozygous G13830A mutation in AT gene.</p>

The morphological and immunological characteristics of less-differentiated acute myeloid leukemic cells / 中国实验血液学杂志

The aim was to study the morphological, histochemical and immunological characteristics of less-differentiated acute myeloid leukemic cells, and their diagnostic significance. Wright-Giemsa and histochemical staining were used to stain bone marrow smears from 2 case of AML-Mo. Immunological phenotypes were determined with flow cytometry. The results showed that myeloperoxidase stainings of both cases were negative, PAS was positive with fine particles, CD33/CD13 were positive, CD2/CD3/CD10/CD19/CD22 were negative. It is concluded that morphology, histochemistry and immunological phenotype on bone marrow smears are the main diagnostic basis for AML-Mo. The use of multiple monoclonal antibodies for staining may improve the accuracy.