Establishment of leukemia cell model with inducible AML1-ETO expression and its effect on fatty acid metabolism in leukemia cells / 中华血液学杂志

Objective: To investigate the effect of the AML1-ETO (AE) fusion gene on the biological function of U937 leukemia cells by establishing a leukemia cell model that induces AE fusion gene expression. Methods: The doxycycline (Dox) -dependent expression of the AE fusion gene in the U937 cell line (U937-AE) were established using a lentivirus vector system. The Cell Counting Kit 8 methods, including the PI and sidanilide induction, were used to detect cell proliferation, cell cycle-induced differentiation assays, respectively. The effect of the AE fusion gene on the biological function of U937-AE cells was preliminarily explored using transcriptome sequencing and metabonomic sequencing. Results: ①The Dox-dependent Tet-on regulatory system was successfully constructed to regulate the stable AE fusion gene expression in U937-AE cells. ②Cell proliferation slowed down and the cell proliferation rate with AE expression (3.47±0.07) was lower than AE non-expression (3.86 ± 0.05) after inducing the AE fusion gene expression for 24 h (P<0.05). The proportion of cells in the G(0)/G(1) phase in the cell cycle increased, with AE expression [ (63.45±3.10) %) ] was higher than AE non-expression [ (41.36± 9.56) %] (P<0.05). The proportion of cells expressing CD13 and CD14 decreased with the expression of AE. The AE negative group is significantly higher than the AE positive group (P<0.05). ③The enrichment analysis of the transcriptome sequencing gene set revealed significantly enriched quiescence, nuclear factor kappa-light-chain-enhancer of activated B cells, interferon-α/γ, and other inflammatory response and immune regulation signals after AE expression. ④Disorder of fatty acid metabolism of U937-AE cells occurred under the influence of AE. The concentration of the medium and short-chain fatty acid acylcarnitine metabolites decreased in cells with AE expressing, propionyl L-carnitine, wherein those with AE expression (0.46±0.13) were lower than those with AE non-expression (1.00±0.27) (P<0.05). The metabolite concentration of some long-chain fatty acid acylcarnitine increased in cells with AE expressing tetradecanoyl carnitine, wherein those with AE expression (1.26±0.01) were higher than those with AE non-expression (1.00±0.05) (P<0.05) . Conclusion: This study successfully established a leukemia cell model that can induce AE expression. The AE expression blocked the cell cycle and inhibited cell differentiation. The gene sets related to the inflammatory reactions was significantly enriched in U937-AE cells that express AE, and fatty acid metabolism was disordered.

Preparation of CD52-targeted chimeric antigen receptor-modified T cells and their anti-leukemia effects / 中华血液学杂志

Objective: To construct chimeric antigen receptor (CAR) T cells targeting CD52 (CD52 CAR-T) and validate the effect of CD52 CAR-T cells on CD52-positive leukemia. Methods: A second-generation CD52-targeting CAR bearing 4-1BB costimulatory domain was ligated into a lentiviral vector through molecular cloning. Lentivirus was prepared and packaged by 293 T cells with a four-plasmid system. Fluorescein was used to label cell surface antigens to evaluate the phenotype of CD52 CAR-T cells after infection. Flow cytometry and ELISA were used to evaluate the specific cytotoxicity of CD52 CAR-T cells to CD52-positive cell lines in vitro. Results: ①A pCDH-CD52scFv-CD8α-4-1BB-CD3ζ-GFP expressing plasmid was successfully constructed and used to transduce T cells expressing a novel CD52-targeting CAR. ②On day 6, CD52-positive T cells were almost killed by CD52-targeted CAR-T post lentivirus transduction [CD52 CAR-T (4.48 ± 4.99) %, vs Vector-T (56.58±19.8) %, P=0.011]. ③T cells transduced with the CAR targeting CD52 showed low levels of apoptosis and could be expanded long-term ex vivo. ④The CD52 CAR could promote T cell differentiation into central and effector memory T cells, whereas the proportion of T cells with a CD45RA(+) effector memory phenotype were reduced. ⑤CD52 CAR-T cells could specifically kill CD52-positive HuT78-19t cells but had no killing effect on CD52-negative MOLT4-19t cells. For CD52 CAR-T cells, the percentage of residual of HuT78-19t cells was (2.66±1.60) % at an the E:T ratio of 1∶1 for 24 h, while (56.66±5.74) % of MOLT4-19t cells survived (P<0.001) . ⑥The results of a degranulation experiment confirmed that HuT78-19t cells significantly activated CD52 CAR-T cells but not MOLT4-19t cells[ (57.34±11.25) % vs (13.06± 4.23) %, P<0.001]. ⑦CD52 CAR-T cells released more cytokines when co-cultured with HuT78-19t cells than that of vector-T cells [IFN-γ: (3706±226) pg/ml, P<0.001; TNF-α: (1732±560) pg/ml, P<0.01]. Conclusions: We successfully prepared CD52 CAR-T cells with anti-leukemia effects, which might provide the foundation for further immunotherapy.

Preparation of CD33 targeted bispecific- and trispecific-T cell engagers and their cytotoxicity on leukemia cells / 中华血液学杂志

Objective: To investigate the effect of CD33-targeted bi-specific and tri-specific T-cell engagers on T-cell proliferation and explore their cytotoxicity on leukemia cells. Methods: The CD33-targeted bi-specific T-cell engager (CD33-BiTE) and tri-specific T-cell engager (CD33-TriTE) expression vectors were successfully constructed and expressed through a eukaryotic cell expression system. CD33-BiTE and CD33-TriTE were purified by affinity chromatography. The effects of CD33-BiTE and CD33-TriTE on T cells were analyzed through in vitro experiments. Results: ① CD33-BiTE and CD33-TriTE were successfully constructed and purified and could compete with flow cytometry antibodies for binding to the target cells. ② After 12 days of co-culture with CD33-BiTE and CD33-TriTE, the number of human T cells were expanded to 33.89±19.46 and 81.56±23.62 folds, respectively. CD33-TriTE induced a stronger proliferation of T cells than CD33-BiTE (P<0.05) . ③ Both CD33-BiTE and CD33-TriTE induced specific dose-dependent cytotoxicity on CD33(+) leukemia cells. ④ Compared to CD33-TriTE, leukemia cells were prone to express PD-L1 when co-cultured with T cells and CD33-BiTE. CD33-TriTE induced powerful cytotoxicity on leukemia cells with high PD-L1 expression. Conclusion: CD33-BiTE and CD33-TriTE expression vectors were constructed, and fusion proteins were expressed in eukaryotic cells. Our results support the proliferative and activating effects of BiTE and TriTE on T cells. Compared to that of CD33-BiTE, CD33-TriTE induced a stronger proliferative effect on T cells and a more powerful cytotoxicity on leukemia cells with high PD-L1 expression.

Effects of interferon regulatory factor 9 on the biological phenotypes in PML-RARα-induced promyelocytic leukemia / 中华血液学杂志

Objective: To investigate the prognostic significance of interferon regulatory factor 9 (IRF9) expression and identify its role as a potential therapeutic target in acute promyelocytic leukemia (APL) . Methods: The gene expression profile and survival data applied in the bioinformatic analysis were obtained from The Cancer Genome Atlas and Beat acute myeloid leukemia (AML) cohorts. A dox-induced lentiviral system was used to induce the expression of PML-RARα (PR) in U937 cells, and the expression level of IRF9 in U937 cells treated with or without ATRA was examined. We then induced the expression of IRF9 in NB4, a promyelocytic leukemia cell line. In vitro studies focused on leukemic phenotypes triggered by IRF9 expression. Results: ①Bioinformatic analysis of the public database demonstrated the lowest expression of IRF9 in APL among all subtypes of AML, with lower expression associated with worse prognosis. ②We successfully established a PR-expression-inducible U937 cell line and found that IRF9 was downregulated by the PR fusion gene in APL, with undetectable expression in NB4 promyelocytic cells. ③An IRF9-inducible NB4 cell line was successfully established. The inducible expression of IRF9 promoted the differentiation of NB4 cells and had a synergistic effect with lower doses of ATRA. In addition, the inducible expression of IRF9 significantly reduced the colony formation capacity of NB4 cells. Conclusion: In this study, we found that the inducible expression of PR downregulates IRF9 and can be reversed by ATRA, suggesting a specific regulatory relationship between IRF9 and the PR fusion gene. The induction of IRF9 expression in NB4 cells can promote cell differentiation as well as reduce the colony forming ability of leukemia cells, implying an anti-leukemia effect for IRF9, which lays a biological foundation for IRF9 as a potential target for the treatment of APL.

Functional investigation of chimeric antigen receptor T cells targeting LMP1 antigen / 中华血液学杂志

Objective: This study aimed to create a type of CAR-T cells that targets LMP1 antigen and study its immunotherapeutic effect on LMP1-positive hematological malignancies. Methods: To generate LMP1 CAR-T cells, a plasmid expressing LMP1 CAR was created using molecular cloning technology, and T cells were infected with LMP1 CAR lentivirus. The effects of LMP1 CAR-T cells on specific cytotoxicity against LMP1-positive tumor cell lines infected with the EB virus had been confirmed. Results: ① LMP1 protein expressing on EB virus-positive lymphoma cells surface was verified. ② The LMP1 CAR-expressing plasmid was created, and LMP1 CAR-T cells were obtained by infecting T cells with a lentivirus packaging system, with an infection efficiency of more than 80% . ③LMP1 CAR-T cells have a 4∶1 effect-to-target ratio in killing LMP1-positive lymphoma cells. The killing effect of LMP1 CAR-T cells on Raji cells was enhanced after 48 h of coculture, but there was no significant killing effect on Ramos, which are LMP1-negative lymphoma cells. ④After coculture with LMP1-positive lymphoma cells at a ratio of 1∶1 for 5 h, the degranulation effect was enhanced. The proportion of CD107a(+) T cells in the LMP1 CAR-T cell treatment group was significantly higher than that in the vector-T cell group [ (13.25±2.94) % vs (1.55±0.05) % , t=3.972, P=0.017]. ⑤After coculture with LMP1-positive lymphoma cells, the proportion of CD69(+) and CD25(+) T cells in the LMP1 CAR-T cell group was significantly higher than that in vector-T cell group [ (7.40±0.41) % vs (3.48±0.47) % , t=6.268, P=0.003; (73.00±4.73) % vs (57.67±2.60) % , t=2.842, P=0.047]. ⑥After coculture with LMP1-positive lymphoma cells, cytokine secretion in the LMP1 CAR-T cell group was higher than that in the vector-T cell group [interferon-gamma: (703±73) ng/L vs (422±87) ng/L, t=2.478, P=0.068; tumor necrosis factor-alpha: (215±35) ng/L vs (125±2) ng/L, t=2.536, P=0.064]. Conclusion: In this study, we found that the LMP1 protein is only found on the surface of the EBV-positive tumor cell. Simultaneously, we created an LMP1 CAR-expressing plasmid and obtained LMP1 CAR-T cells by infecting T cells with a lentivirus packaging system. Furthermore, we demonstrated that LMP1 CAR-T cells could specifically kill LMP1-positive tumor cells in vitro. The degranulation and activation effects of LMP1 CAR-T cells were enhanced after coculture with LMP1-positive tumor cells, indicating a potential clinical application.

Germacranolide sesquiterpenes from Carpesium cernuum and their anti-leukemia activity / 中国天然药物

In this study, three new germacranolide sesquiterpenes (1-3), together with six related known analogues (4-9) were isolated from the whole plant of Carpesium cernuum. Their structures were established by a combination of extensive NMR spectroscopic analysis, HR-ESIMS data, and ECD calculations. The anti-leukemia activities of all compounds towards three cell lines (HEL, KG-1a, and K562) were evaluated in vitro. Compounds 1-3 exhibited moderate cytotoxicity with IC

Distribution of ETV6-ABL Fusion Gene in Hematopoietic Cell Populations of Myeloproliferative Neoplasm / 中国实验血液学杂志

OBJECTIVE@#To explore the expression level of ETV6-ABL fusion gene in different cell populations in patients with myeloproliferative neoplasm (MPN) and therapeutic effect of tyrosine kinase inhibitor (TKI).@*METHODS@#A 42-year-old man who presented with fever, marked leukocytosis and chronic myelogenous leukemia (CML) like MPN was reported. ETV6-ABL fusion gene was detected by real-time PCR and confirmed by direct sequencing. ETV6-ABL mRNA expression in each cell population sorted from peripheral blood by flow cytometry was detected by real-time PCR.@*RESULTS@#ETV6-ABL fusion gene was found out in bone marrow cells and confirmed as type A by direct sequencing. ETV6-ABL fusion gene transcript level in polymorphonuclear cells was nearly 3.6-fold relative to that in total cells, which was significantly higher than that in T cell, B cell and monocyte subsets. The complete blood count (CBC) returned to normal level after treatment with imatinib (400 mg) daily for three months. After TKI treatment for 6 months, the ratio of ETV6-ABL/ABL decreased from 174.1% to 1.9%.@*CONCLUSION@#ETV6-ABL fusion gene positive MPN may have a CML clinical presentation and is sensitive to TKI. ETV6-ABL fusion gene is specifically expressed in polymorphonuclear cells.

Effect and Mechanism of Midostaurin Derivatives 5'''-methoxyfradcarbazole A on Mouse Leukemia CB3 Cells / 中国药学杂志

OBJECTIVE: To investigate the mechanism of midostaurin derivative 5'''-methoxyfradcarbazole A of action in inhibiting mouse leukemia cells (CB3) growth. METHODS: MTT assay was employed to evaluate the effect of compound 5'''-methoxyfradcarbazole A on the proliferation of CB3 cells, and generate the growth inhibition curves. Flow cytometry and Annexin V-FITC /PI double staining were used to determine the changes of the cell cycle, cell differentiation and apoptosis. Western blot analysis was applied to test the effects of 5'''-methoxyfradcarbazole A on cyclin and apoptosis-related proteins. RESULTS: The compound 5'''-methoxyfradcarbazole A could significantly inhibit the growth of CB3 cells, and the half maximal inhibitory concentration (IC50) of the compound was (0.587±0.135)μmol•L-1. 5'''-Methoxyfradcarbazole A was able to induce early apoptosis and late apoptosis of CB3 cells in a time- and dose-dependent manner. At the same time, it also affected the cell cycle of CB3 and significantly increased the proportion of G2 phase. The expression of CD41, a platelet differentiation marker, and Ter119, an erythrocyte differentiation marker, were also increased in CB3 cells treated with 5'''-methoxyfradcarbazole A. Besides, the expressions of apoptosis-related proteins Bim, PARP1 and cyclin-related protein P21 were significantly increased, and the phosphorylated ERK protein was decreased. CONCLUSION: Midostaurin derivative 5'''-methoxyfradcarbazole A could increase the apoptosis of CB3 cells by promoting the expression of apoptotic protein Bim, and cause G2 phase arrest by increasing the expression of cyclin protein P21. Also, the changes of the expression levels of PARP1 and phosphorylated ERK protein also partly explain the inhibition of 5'''-methoxyfradcarbazole A on CB3 cells growth.

Extract of Naotaifang, a compound Chinese herbal medicine, protects neuron ferroptosis induced by acute cerebral ischemia in rats / 中西医结合学报

OBJECTIVE@#Our previous research showed that Naotaifang (a compound traditional Chinese herbal medicine) extract (NTE) has clinically beneficial effects on neurological improvement of patients with acute cerebral ischemia. In this study, we investigated whether NTE protected acute brain injury in rats and whether its effects on ferroptosis could be linked to the dysfunction of glutathione peroxidase 4 (GPX4) and iron metabolism.@*METHODS@#We established an acute brain injury model of middle cerebral artery occlusion (MCAO) in rats, in which we could observe the accumulation of iron in neurons, as detected by Perl's staining. Using assay kits, we measured expression levels of ferroptosis biomarkers, such as iron, glutathione (GSH), reactive oxygen species (ROS) and malonaldehyde (MDA); further the expression levels of transferrin receptor 1 (TFR1), divalent metal transporter 1 (DMT1), solute carrier family 7 member 11 (SLC7A11) and GPX4 were determined using immunohistochemical analysis, real-time quantitative polymerase chain reaction and Western blot assays.@*RESULTS@#We found that treatment with NTE reduced the expression levels of TFR1 and DMT1, reduced ROS, MDA and iron accumulation and reduced neurobehavioral scores, relative to untreated MCAO rats. Treatment with NTE increased the expression levels of SLC7A11, GPX4 and GSH, and the number of Nissl bodies in the MCAO rats.@*CONCLUSION@#Taken together, our data suggest that acute cerebral ischemia induces neuronal ferroptosis and the effects of treating MCAO rats with NTE involved inhibition of ferroptosis through the TFR1/DMT1 and SCL7A11/GPX4 pathways.

Targeting BCMA in multiple myeloma using chimeric antigen receptor-engineered T cells / 中华血液学杂志

Objective@#To construct the BCMA-CAR using the B-cell maturation antigen (BCMA) specific ligand APRIL as antigen binding region and to validate the effect of BCMA-CAR modified T cells (BCMA-CAR-T) on myeloma cells.@*Methods@#The BCMA-CAR was constructed using the BCMA specific ligand APRIL as antigen binding domain and 4-1BB as the costimulatory domain. The specific cytotoxicity against BCMA+ myeloma cell lines and primary multiple myeloma (MM) cells in vitro were evaluated. In addition, BCMA+ myeloma xenograft mouse model was established to assess the anti-tumor effect of BCMA-CAR-T cell therapy in vivo.@*Results@#BCMA-CAR-T cells could specifically kill BCMA+ myeloma cell lines (For BCMA-CAR-T cells, BCMA+ cells are almost undetectable in the E∶T ratio of 1∶4) and MM patients’ bone marrow mononuclear cells (the proportion of residual cells in BCMA-CAR-T and vector-T groups was 16.0% vs 66.85%, P=0.003) with significant degranulation (CAR-T and vector-T cells cocultured with MM1.S, H929 and U266 had degranulation levels of 33.30% vs 5.62%, 16.97% vs 2.95% and 25.87% vs 2.97%, respectively, P<0.001) and cytokines release (P<0.01) in vitro. In a human BCMA+ myeloma xenograft mouse model, BCMA-CAR-T cells could significantly prolong the survival of mice (The median survival time of mice treated with BCMA-CAR-T and vector-T cells was 87.5 days and 67.5 days, respectively, P<0.001) .@*Conclusion@#The ligand-based BCMA-CAR-T cells could be a promising strategy for BCMA+ multiple myeloma treatment.

Effect of Chemotherapeutic Drug-Induced Damage of Bone Marrow Stroma Cells on Normal Hematopoietic Cells / 中国实验血液学杂志

OBJECTIVE@#To explore the effect of damage of bone marrow stroma cells induced by chemotherapeutic drug on the function of normal hematopoitic cells.@*METHODS@#Senescence cells were detected by flow cytometry after SA-β-gal staining; real-time PCR was used to detect the expression of a serial molecules in bone marrow stromal cell line OP9 cells; the expression of γ-H2AX was determined by flow cytometry after histone γ-H2AX staining; the colony forming ability of hematopoietic cells was tested by colony formation assay.@*RESULTS@#The percentage of senescence cells in OP9 cells after DNR treatment was 2.24 times as much as that in untreated OP9 cells (P<0.05). Compared with normal OP9 cells, the expression levels of IL-6 and TNF-alpha in DNR-treated OP9 cells increased by 2.73 times (P<0.01) and 0.56 times (P<0.01), and the expression levels of N-cadherin, alpha smooth muscle actin (alpha-SMA), angiopoietin1 (Angpt1) and osteopontin (OPN) decreased by 69.54%（P<0.01），63.90%（P<0.01），87.41%（P<0.01）and 42.78%（P<0.01）respectively. After the co-culture with DNR-treated OP9 cells, the colony formation of normal hematopoietic cells decreased by 47.10% than that co-cultured with untreated OP9 cells (P< 0.05), meanwhile, the percentage of γ-H2AX+ cells in normal hematopoietic cells increased by 2.19 times (P<0.05).@*CONCLUSION@#After treatment with DNR, the senescence cell number of OP9 cells sgnificantly increases; the expression of TNF-α and IL-6 is up-regulated, while the expression of α-SMA, Angpt-1 and OPN is down-regulated as compared with normal OP9 cells. In addition, after co-culture of DNR-treated OP9 cells with normal hematopoietic cells, the colony formation ability of hematopoietic cells decreases and the genome instability of hematopoietic cells increases as compared with normal hematopoietic cells.

Oxidative Damage of Bone Marrow Stromal Cells Caused by Chemotherapy Drugs / 中国实验血液学杂志

OBJECTIVE@#To explore the oxidative damage of OP9 cells induced by daunorubicin (DNR) treatment.@*METHODS@#The TMRM probe was used to detect mitochondrial membrane potential by flow cytometry; the reactive oxygen species (ROS) was determined by flow cytometry DCFDA probe; the real-time PCR was used to detect the molecular expression of antioxidant enzyme，glutathione peroxidase (GPX) in OP9 cells; the expression of γ-H2AX was determined by flow cytometry.@*RESULTS@#Compared with normal OP9 cells, the positive rate of TMRM in DNR-treated OP9 cells decreased by 56.7% (P＜0.05); the positive rate of DCFDA in DNR-treated OP9 cells increased by 3.52 times (P＜0.01). Compared with normal OP9 cells, DNR-treated OP9 cells showed a decrease in the expression of GPX4 by 44.22% (P＜0.001); the expression of GPX7 decreased by 65.7% (P＜0.001); the expression of GPX8 decreased by 24.7% (P＜0.001); the positive rate of γ-H2AX in DNR-treated OP9 cells increased (P＜0.05).@*CONCLUSION@#After DNR treatment, mitochondrial membrane potential of OP9 cells decreases; the level of reactive oxygen species increases; the expression of glutathione peroxidase (GPX) molecules decreases significantly; genomic instability increases obviously; the oxidative damage of cells increased.

Impact of collection methods and storage conditions on the recovery of tear proteins / 国际眼科杂志(Guoji Yanke Zazhi)

AIM: To explore the optimal ways for the collection and preservation of tear proteins.? METHODS: Siliconized microcapillary tube or Schirmer's strips made in China or in the USA were tested. All three carriers were loaded with equal volume and quantity of bull serum albumin (BSA), fetal bovine serum (FBS) or known concentrations of IL1-Ra, IL-6, MIG, IP-10, MMP9, TIMP1, VEGF and HBD1 mixture and kept at 4℃, -20℃ or -80℃. After a desired time of storage, the proteins were eluted with different elution buffer. Micro BCATM protein quantitative kit and enzyme-linked immunosorbent assay kits were used to determine total protein concentrations and the concentrations of the above biological molecules, respectively.?RESULTS: Neither protease inhibitors nor detergent were necessary for protein elution from the Schirmer's strip. When stored at-20℃ and-80℃, three tear carriers showed no differences in the recovery of total protein. The recovery of IL-6 from three carriers under different temperature was similar but the higher than other proteins. The recovery rate of IL-1Ra was 98％±6％ from the microcapillary tube and 19％± 15％ from Schirmer's strip. The recovery rate of HBD1 was 93％± 8％ from the microcapillary tube and 61％± 3％ from Schirmer's strip. The recovery rate of MIG was 87％ ± 4％ from the microcapillary tube and 69％± 4％ from Schirmer's strip. Microcapillary tube also had less retention for MMP-9 ( 90％ ± 5％) and TIMP1 ( 103％ ± 7％) compared to Schirmer's strip(62％±3％ vs 87％±5％). The strips made in China and in the USA showed similar retention to most of the proteins tested here. When stored at -80℃, the recovery rate for VEGF and IP-10 were 82％± 5％ and 72％± 8％, respectively, significantly higher than that when stored at higher temperature.?CONCLUSION: The selection of tear sample carrier is dependent on target proteins to be recovered. Low temperature deep freezing is a preferred way for tear sample storage. microcapillary tube is applicable for most of the proteins. While for those protein with noticeable polarity, like HBD1, pre - experiment is needed.

Exploration of Mechanism for Meisoindigo-Inducing K562 Cell Apoptosis by Using Quantitative Proteomic Analysis / 中国实验血液学杂志

OBJECTIVE@#To screen the differentially expressed proteins at the early stage of K562 cells treated with meisoindigo by using tandem mass tags (TMT)-based proteomics technology, and to explore the mechanism for meisoindigo-inducing apoptosis.@*METHODS@#The half inhibitory concentration (IC) of mesoindigo on K562 cells was determined by CCK8. The flow cytometry was used to assay the apoptosis of K562 cells treated by meisoindigo or DMSO. Total proteins were extracted from the cells treated with 0.2% DMSO (control) or 20 μmol/L meisoindigo (Test) for 2 hours. Then, the TMT-labeling HPLC-MS/MS was used to identify and quantify the peptides and their abundance, all the tests were repeated for 3 times. The Mascot software was used to identify the proteins; the GO annotations, enrichment and cluster analysis were used to analyze the differentially expressed proteins.@*RESULTS@#Meisoindigo-induced K562 cell apoptosis in a dose-dependent manner (r=0.98), 5 544 proteins were identified, 4792 of which were quantified. The protein with expression difference>1.5-folds in Test group accoanted for 8, out of which the expression of 4 proteins were up-regulated and 4 were down-regulated. The differentially expressed proteins mainly associated with reactive oxygen species (ROS).@*CONCLUSION@#Several proteins including DDIT4 were found to have dramatic changes in the early stage of K562 cells treated with meisoindigo by using quantitative proteomics technology. The ROS metabolic process may play important roles in meisoindigo-inducing apoptosis of K562 cells.

Study of the effects and mechanism of all-trans retinoic acid on leukemic cell line U937 cells with NPM1 mutation / 中华血液学杂志

Objective@#To investigate the effect and mechanism of all-trans retinoic acid (ATRA) on leukemic cell line U937 cells with NPM1 mutation.@*Methods@#Human acute myeloid leukemia cell line U937 was explored, NPM1 mutated (A type) plasmids were transfected into U937 to form stable clones A1 and A2, which were identified by Western blot and Co-immunoprecipitation. The cell proliferation was measured by methylthiazolyl tetrazolium bromide (MTT) ; cell cycle and cell apoptosis were explored by flow cytometric; cell colony formation was measured by microscope count, the molecular pathways related to cell proliferation were measured by Western blot.@*Results@#①The cell proliferations of mutant A1 and A2 were inhibited significantly by 52.6% and 35.8% (P<0.05) , respectively under ATRA exposure. ②The percentages of G0/G1 stage of mutant A1 and A2 increased by 20.1% and 35.8%, respectively under ATRA exposure. ③All the U937 leukemic cells were inhibited under ATRA exposure; the decreased percentages of vector, wild-type and mutant NPM1 cells were 32.7%, 57.9% and 90.9% respectively. ④p-ERK decreased obviously after ATRA exposure in NPM1 mutated leukemic cells. ⑤More mutant NPM1 cells inclined to apoptosis under the exposure of ATRA and cytotoxic drugs than cytotoxic drugs alone, meanwhile more cells apoptosis occurred when ATRA was administrated after cytotoxic drugs exposure.@*Conclusions@#ATRA could inhibit cell proliferation and colony formation, blocked the cell cycle in the G0/G1 stage accompanied by the significant reduction of p-ERK in U937 leukemic cells with NPM1 mutation. Besides, ATRA could synergize with drugs to suppress the leukemic cells survival more effectively when ATRA was administered after the cytotoxic drugs exposure in U937 leukemic cells with NPM1 mutation.

Expression and Asymmetric Division of Numb in Leukemia Cell K562 Line / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate the role of asymmetric division in leukemia cells through detection of expression and asymmetric division of Numb in differentiated and undifferentiated K562 cells.</p><p><b>METHODS</b>Firstly, Hemin was used to induce K562 cell differentiation, and the expression of Numb was detected by the real-time quantitative RT-PCR and flow cytometry. After K562 cells were synchronized by nocodazole, the Numb protein was labeled by immunohistochemical staining, followed by the determination of the terminally differentiated cells through confocal microscopy. The fluorescence intensity was calculated by Image J software, and the cell division pattern was analyzed on the basis of the fluorescence intensities of Numb in 2 divided daughter cells.</p><p><b>RESULTS</b>Compared with the undifferentiated K562 cells, the level of Numb mRNA expression increased 2.3 times (P<0.001). The ratio of Numb positive cells was(67.37±5.01)% in differentiated K562 cells, while that was (43.97±5.72)% in undifferentiated K562 cells (P<0.01). Compared with undifferentiated K562 cells, the ratio of cells with asymmetric division in differentiated K562 cells increased 18.3%, the percentage of cells with symmetry self-renewal reduced 49.7%(P<0.001) and that with symmetry differentiation increased 32%(P<0.001).</p><p><b>CONCLUSION</b>In differentiated K562 cells, expression of Numb and proportion of cells with asymmetric division were higher than that in undifferentiated cells. With the differentiation of leukemia cells, the proportion of cells with asymmetrical division increases, and the proportion of cells with symmetrical self-renewal decreases. The stemness of leukemia cells is maintained mainly through the symmetrical self-renewal.</p>

Relationship between High Expression of c-FLIP and Drug Resistance of Leukemia Cells / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To explore the effect of c-FLIP expression on drug resistance of Kasumi-1 leukemia cells and its mechanisms.</p><p><b>METHODS</b>Tet-on inducible system was used to construct the conditional expression vector of c-FLIP by cloning the c-FLIP gene into lentivirus vector pLVX-Tight-Puro, then the Kasumi-1 cells were transfected with lentivirus pLVX-Tight-Puro-c-FLIP. The expression of c-FLIP was induced by doxycycline(Dox) for different time and doses, and verified by qRT-PCR and Western blot. On the basis of the overexpression of c-FLIP, the Kasumi-1-c-FLIP cells were treated with CH11 and PB in order to induce apoptosis, and the Giemsa staining was used to show the apoptotic cell morphology.</p><p><b>RESULTS</b>qRT-PCR and Western blot showed the overexpression of c-FLIP, the CH11 and PB can induce Kasumi-1 cell apoptosis, while the c-FLIP overexpression weakened this effects. Western blot showed that the c-FLIP blocked the caspase-8 activation.</p><p><b>CONCLUSION</b>The overexpression of c-FLIP inhibits the apoptosis caused by CH11 and PB, and leads to drug resistance in leukemia cells.</p>

Reconstruction of Humanized Bone Marrow Niche in Immunodeficiency Mouse / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To reconstruct a human bone marrow niche in immunodeficiency mouse (NOD/SCID) so as to provide a model for observing the effect of abnormal BM niche on the occurence and development of leukemia.</p><p><b>METHODS</b>Human platelet lysate(HPL) was obtained by repeated freezing and thawing of concentrated platelet. Bone marrow-derived mesenchymal stem cells were cultured in α-minimal essential medium (α-MEM) containing 10% HPL or 10% FBS. The morphology, cell phenotype, multilineage differentiation potential in vitro and proliferation capacity between the mesenchymal stem cells cultured with HPL or FBS were compared. The human bone marrow formation capacity of HPL-cultured MSC was observed. The MSC was seeded on β-TCP scaffolds for 12h, then the MSC-coated scaffold were implanted in a subcutaneous pocket on the dorsum of NOD/SCID mice. After 8-12 week, the scaffolds were harvested from the mice, then fixed, paraffin-embedded and stained for HE.</p><p><b>RESULTS</b>Whether cultured in the presence of HPL or FBS, the MSC all displayed a spindle-shaped fibroblast-like morphology; the flow cytometry analysis revealed no obvious differences in cell immunophenotype in this 2 groups; they all have the ability to differentiate towards osteoblasts, adipocytes, and chondrocytes in vitro. However, the mesenchymal stem cells cultivated with HPL-contained medium showed stronger proliferation capacity and higher activity to differentiate towards osteoblasts. Mesenchymal stem cells cultivated with HPL still have in vivo bone-forming capacity.</p><p><b>CONCLUSION</b>HPL cultured MSC have stronger proliferation capacity and potential of differentiate towards osteoblasts, HPL-cultured MSC also can reconstruct humanized bone marrow niche in murine host.</p>

Effect of TBLR1-RARα Fusion Gene on Erythroid Differentiation of K562 Cells / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To explore the effects of TBLR1-RARα on the differentiation induction of leukemia cell line K562 cells into erythroid lineage and to investigate its related mechanisms.</p><p><b>METHODS</b>Tet-Off inducible system was used to construct the conditional expression vector of TBLR1-RARα fusion gene by cloning the TBLR1-RARα fragment into lentivirus vector pLVX-Tight-Puro, the expression of TBLR1-RARα fusion gene was induced by doxycycline (Dox). Then, K562 cells were transfected with lentivirus pLVX-Tight-Puro-TBLR1-RARα-flag, and the expression of fusion proteins was verified by Western blot. After treatment of K562 with all-trans retinoid acid (ATRA), real time RT-PCR was performed to test the expression of erythroid differentiation-related CD71 and α, ε, γ-globins gene. Flow cytometry was used also to analyze the expression of erythroid differentiation markers CD71 and CD235a. Benzidine staining was used to detect the production of hemoglobin in K562 cells.</p><p><b>RESULTS</b>qRT-RCR showed that ATRA could increase the expression level of CD71 and α, ε, γ-globin genes when TBLR1-RARα was expressed. After treatment of ATRA, the proportion of CD71(+) cells detected by the flow cytometry also increased. Benzidine staining showed that ATRA could induce hemoglobin production in K562 cells with TBLR1-RARα fusion gene expression.</p><p><b>CONCLUSION</b>The expression of TBLR1-RARα fusion gene contribute to ATRA-inducing differentiation of K562 cells into erythroid lineage.</p>

Clinical characteristics of newly diagnosed acute myeloid leukemia patients with NPM1 mutation / 中国实验血液学杂志

The purpose of this study was to investigate the clinical characteristics of newly diagnosed acute myeloid leukemia (AML) patients with NPM1 mutation in exon 12 and to explore the relationship between NPM1 mutation and FLT3-ITD, IDH1 mutation. The AML clinical data and bone marrow samples of patients were collected. The diagnosis and classification were based on WHO criteria. The genomic DNA was extracted and NPM1 mutation was detected by sequencing after PCR. The specimens of 389 AML patients were tested. The results showed that the NPM1 mutation was found in 14.1% samples (55/389). The incidence of FLT3-ITD mutation was 14.7% (57/389) . The incidence of IDH1 mutation was 6.4% (25/389) . NPM1 mutation was not detected in AML with AML1-ETO, PML-RARA or CBF-MYH11 fusion genes. The incidences of FLT3-ITD and IDH1 mutation were 29.1% and 12.7% respectively in AML with NPM1 mutation. The incidences of FLT3-ITD and IDH1 mutation were 12.3% and 5.4% respectively in AML without NPM1 mutation. The incidences of FLT3-ITD and IDH1 mutation were significantly higher in AML with NPM1 mutation than that in AML without NPM1 mutation. The incidence of NPM1 mutation in normal karyotype AML was 26.5% (35/132) which significantly higher than that in other AML. The AML with NPM1 mutation characterized by older age, high platelet number, higher incidence in AML-M5, lower CD34 positive cells, more possible co-existence with FLT3-ITD and IDH1 mutation and other clinical features. The complete remission rate after one cycle of induction chemotherapy was 69.8% in AML without NPM1 mutation. The complete remission rate after one cycle of induction chemotherapy was 72.2% in AML with NPM1 mutation, there was no significant difference between them (P = 0.07). It is concluded that AML with NPM1 mutation has distinct clinical features. NPM1 mutation can co-exists with FLT3-ITD and IDH1 mutation, but not with AML1-ETO, PML-RARA or CBF-MYH11 fusion genes.