Transcriptome and Regulatory Network Analyses of CD19-CAR-T Immunotherapy for B-ALL / 基因组蛋白质组与生物信息学报·英文版

Chimeric antigen receptor (CAR) T cell therapy has exhibited dramatic anti-tumor efficacy in clinical trials. In this study, we reported the transcriptome profiles of bone marrow cells in four B cell acute lymphoblastic leukemia (B-ALL) patients before and after CD19-specific CAR-T therapy. CD19-CAR-T therapy remarkably reduced the number of leukemia cells, and three patients achieved bone marrow remission (minimal residual disease negative). The efficacy of CD19-CAR-T therapy on B-ALL was positively correlated with the abundance of CAR and immune cell subpopulations, e.g., CD8 T cells and natural killer (NK) cells, in the bone marrow. Additionally, CD19-CAR-T therapy mainly influenced the expression of genes linked to cell cycle and immune response pathways, including the NK cell mediated cytotoxicity and NOD-like receptor signaling pathways. The regulatory network analyses revealed that microRNAs (e.g., miR-148a-3p and miR-375), acting as oncogenes or tumor suppressors, could regulate the crosstalk between the genes encoding transcription factors (TFs; e.g., JUN and FOS) and histones (e.g., HIST1H4A and HIST2H4A) involved in CD19-CAR-T therapy. Furthermore, many long non-coding RNAs showed a high degree of co-expression with TFs or histones (e.g., FOS and HIST1H4B) and were associated with immune processes. These transcriptome analyses provided important clues for further understanding the gene expression and related mechanisms underlying the efficacy of CAR-T immunotherapy.

Proteomic changes in a childhood acute lymphoblastic leukemia cell line during the adaptation to vincristine / Cambios en el proteoma de una línea celular de leucemia linfoblástica aguda infantil durante la adaptación a vincristina

Abstract: Introduction: Relapse occurs in approximately 20% of Mexican patients with childhood acute lymphoblastic leukemia (ALL). In this group, chemoresistance may be one of the biggest challenges. An overview of complex cellular processes like drug tolerance can be achieved with proteomic studies. Methods: The B-lineage pediatric ALL cell line CCRF-SB was gradually exposed to the chemotherapeutic vincristine until proliferation was observed at 6 nM, control cells were cultured in the absence of vincristine. The proteome from each group was analyzed by nanoHPLC coupled to an ESI-ion trap mass spectrometer. The identified proteins were grouped into over-represented functional categories with the PANTHER classification system. Results: We found 135 proteins exclusively expressed in the presence of vincristine. The most represented functional categories were: Toll receptor signaling pathway, Ras Pathway, B and T cell activation, CCKR signaling map, cytokine-mediated signaling pathway, and oxidative phosphorylation. Conclusions: Our study indicates that signal transduction and mitochondrial ATP production are essential during adaptation of leukemic cells to vincristine, these processes represent potential therapeutic targets.

Resumen: Introducción: Aproximadamente el 20% de los pacientes mexicanos con leucemia linfoblástica aguda (LLA) infantil presentan recaídas. En este grupo, la quimiorresistencia es uno de los principales desafíos. Los estudios proteómicos pueden dar un panorama general de procesos celulares complejos como la tolerancia a fármacos. Métodos: La línea celular de LLA de linaje B, CCRF-SB, fue expuesta de manera gradual al fármaco quimioterapéutico vincristina hasta observar proliferación celular en presencia de 6 nM, como control se cultivaron células en ausencia del fármaco. Se analizó el proteoma de cada grupo mediante nanoHPLC acoplado a un espectrómetro de masas de tipo trampa de iones. Las proteínas identificadas se agruparon en categorías funcionales sobre-representadas con el sistema de clasificación PANTHER. Resultados: Encontramos 135 proteínas expresadas exclusivamente en presencia de vincristina. Las categorías funcionales más representadas fueron la señalización asociada a los receptores tipo Toll, señalización dependiente de Ras, activación de células B y T, mapa de señalización CCKR, señalización mediada por citoquinas y la fosforilación oxidativa. Conclusiones: Nuestro estudio indica que la transducción de señales y la producción de ATP mitocondrial son procesos esenciales durante la adaptación de células leucémicas a vincristina por lo que estos procesos representan potenciales blancos terapéuticos.

Effects of bufalin on up-regulating methylation of Wilm's tumor 1 gene in human erythroid leukemic cells / 中国结合医学杂志

<p><b>OBJECTIVE</b>To explore the effects of bufalin on inhibiting proliferation, up-regulating methylation of Wilm' tumor 1 gene (WT1) as well as its possible mechanisms in human erythroid leukemic (HEL) cells.</p><p><b>METHODS</b>The HEL cells were treated with bufalin at various concentrations to observe cellular morphology, proliferation assay and cell cycle. The mRNA and protein expression levels of WT1 were detected by reverse transcription polymerase chain reaction (RT-PCR), Western blot and immunocytochemistry, DNA methylation of WT1 and protein expression levels of DNA methyltransferase 3a (DNMT3a) and DNMT3b were analyzed by methylation-specific PCR, and Western blot respectively.</p><p><b>RESULTS</b>The bufalin was effective to inhibit proliferation of HEL cells in a dose-dependent manner, their suppression rates were from 23.4%±2.1% to 87.2%±5.4% with an half maximal inhibit concentration (IC) of 0.046 μmol/L. Typical apoptosis morphology was observed in bufalin-treated HEL cells. The proliferation index of cell cycle decreased from 76.4%±1.9% to 49.7%±1.3%. The expression levels of WT1 mRNA and its protein reduced gradually with increasing doses of bufalin, meanwhile, the methylation status of WT1 gene changed from unmethylated into partially or totally methylated. While, the expression levels of DNMT3a and DNMT3b protein gradually increased by bufalin treatment in a dose-dependent manner.</p><p><b>CONCLUSIONS</b>Bufalin can not only significantly inhibit the proliferation of HEL cells and arrest cell cycle at G/Gphase, but also induce cellular apoptosis and down-regulate the expression level of WT1. Our results provide the evidence of bufalin for anti-leukemia, its mechanism may involve in increasing WT1 methylation status which is related to the up-regulation of DNMT3a and DNMT3b proteins in erythroid leukemic HEL cells.</p>

Grandisin induces apoptosis in leukemic K562 cells

Abstract In this study, the potential antileukemic activity of grandisin, a lignan extracted from Piper solmsianum, was evaluated against the leukemic line K562. The cytotoxicity of grandisin (0.018 to 2.365 µM) was evaluated in K562 and normal peripheral blood lymphocytes by Trypan Blue Exclusion and MTT methods after 48h exposure to the drug. In both methods, cellular viability was concentration-dependent and the IC50 values were lower than 0.85µM. Analysis of K562 cells after treatment with grandisin showed that the cell cycle was arrested in the G1 phase with a 12.31% increase, while both S and G2 phases decreased. Morphological studies conducted after the exposure of K562 to grandisin revealed changes consistent with the apoptosis process, which was confirmed by anexin V stain and caspase activation. Thus, lignan grandisin showed antileukemic activities against the K562 cell line and the cell death process occurred via apoptosis.

Effect of PDK1 on Notch1-Induced Mouse T-cell Acute Lymphoblastic Leukemia / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To explore the role of PDK1 in T-ALL development through establishing the Notch1-induced T-ALL mouse model by using Mx1-cre; LoxP system to knock-out PDK1.</p><p><b>METHODS</b>Cell cycle and apoptosis of leukemic cells were detected by flow cytometry, and relative expression of tumor-related genes and transcription factors of leukemic cells were determined by quantitative real-time PCR.</p><p><b>RESULTS</b>Notch1-induced T-ALL mouse model with inducible knock-out of PDK1 was established successfully. Compared to T-ALL control mouse model, PDK1 knock-out mice showed a significant longer survival time (P<0.01). There was no difference of cell cycle between control and PDK1 knock-out mice, and the apoptosis rate of leukemic cells in PDK1 knock-out mice was higher than that of control mice (P<0.001). PDK1 knock-out resulted in decreased expression of tumor-related genes and transcription factors, such as c-Myc and NF-κB (P<0.01), and increased expression level of P53 (P<0.01).</p><p><b>CONCLUSION</b>PDK1 knock-out can inhibit the development of T-ALL, and its mechanism may be the leukemia progression inhibited by regulating the apoptosis and expression of multiple related genes and transcription factors.</p>

Role of CXCR4/STAT3 in mesenchymal stromal cell-mediated drug resistance of acute leukemia cells / 中华血液学杂志

<p><b>OBJECTIVE</b>To explore the role of CXCR4/STAT3 in mesenchymal stromal cell (MSC)-mediated drug resistance of AML cells.</p><p><b>METHODS</b>AML cell lines U937 and KG1a and primary AML cells were co-cultured with MSC from bone marrow of healthy donors. The AML cell lines cultured alone were used as control. Apoptosis induced by mitoxantrone was measured by flow cytometry. Expression of CXCR4 and STAT3 protein were detected by Western blot. After incubated with STAT3 inhibitor Cucurbitacin I or CXCR4 antagonist AMD3100, the apoptosis of AML cells induced by mitoxantrone was evaluated.</p><p><b>RESULTS</b>Apoptosis of AML cells (U937 and KG1a) and primary AML cells induced by mitoxantrone significantly decreased in cocultured group than that of control group [U937 cells: (20.08±1.53)% vs (45.33 ± 1.03)% , P=0.004; KG1a cells: (25.60 ± 1.82)% vs (40.33 ± 3.29)% , P=0.020]. Expression of phosphorylated STAT3 and CXCR4 protein in AML cells were upregulated in cocultured group. After addition of Cucurbitacin I into the co-culture system, the apoptosis rate of primary AML cells significantly increased. Similar results of the apoptosis rates were also detected when the inhibitor of CXCR4 AMD3100 was added to overcome the stromal cell-mediated drug resistance. Besides, the expression of p-STAT3 in AML cells after incubated with AMD3100 decreased significantly.</p><p><b>CONCLUSIONS</b>AML cells cocultured with MSC leads to the up-regulation of phosphorylated STAT3 and CXCR4 proteins, which resulted in AML cells resistance to chemotherapeutic drugs. Therefore targeting STAT3 or CXCR4 could be a new therapeutic strategy of AML.</p>

Biological mechanisms of human-derived leukemia stem cells senescence regulated by Angelica sinensis polysaccharide / 中国中药杂志

<p><b>OBJECTIVE</b>To explore the biological mechanisms underlying Angelica sindsis polysaccharide (ASP) -induced aging of human-derived leukemia stem cells (LSCs) in vitro.</p><p><b>METHOD</b>Acute myelogenous leukemia stem cells were isolated by magnetic activated cell sorting (MACS). The ability of LSC proliferation treated by various concentration of ASP(20-80 mg · L(-1)) in vitro for 48 hours were tested using cell counting Kit-8 ( CCK8) , colony forming were evaluated by methylcellulose CFU assay. The ultra structure changes of AML CD34+ CD38- cells were analyzed by transmission electron microscopy. The aging cells were detected with senescence-β-galactosidase Kit staining. Expression of aging-related p53, p21, p16, Rb mRNA and P16, Rb, CDK4 and Cyclin E protein were detected by quantitative reverse transcription polymerase chain reaction( qRT-PCR) and Western blotting, respectively.</p><p><b>RESULT</b>The purity of the CD34 + CD38 - cells is (91.15 ± 2.41)% after sorted and showed good morphology. The proliferation of LSC was exhibited significantly concentration-dependent inhibited after exposure to various concentration of ASP. Treated by 40 mg · L(-1) ASP for 48 hours, the percentage of positive cells stained by SA-β-Gal was dramatically increased (P < 0.01) and the colony-formed ability has been weakened (P < 0.01). The observation of ultrastructure showed that cell heterochromatin condensation and fragmentation, mitochondrial swelling, lysosomes increased in number. Aging-related p53, p21, p16, Rb and P16, Rb were up-regulated, protein regulatory cell-cycle CDK4 and Cyclin E were down-regulated. ASP may induce the senescence of LSCs effectively in vitro, P16-Rb cell signaling pathway play a significant role in this process.</p><p><b>CONCLUSION</b>ASP can induce human leukemia stem cell senescence in vitro, the mechanism involved may be related to ASP regulation P16-Rb signaling pathways.</p>

Expression of Notch Gene and Its Role of Anti-apoptosis and Drug-resistance of Cells in Chronic Lymphocytic Leukemia / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate the expression of Notch gene in chronic lymphocytic leukemia cells and to explore the change of Notch protein after the therapy with cytosine arabinoside or dexmethasone, and the mechanism of Notch mediated anti-apoptosis and drug-resistance in chronic lymphocytic leukemia cells.</p><p><b>METHODS</b>The mononuclear cells from bone marrow or peripheral blood of chronic lymphocytic leukemia patients (24 cases) and healthy donors (14 cases) were collected, then the expression of Notch gene, BCL-2, as well as NF-κB gene were detected by real-time fluorescent quantitative PCR (qRT-PCR) at the level of transcription. The change of Notch protein in L1210 cell lines after therapy with cytosine arabinoside and dexmethasone was determined by Western blot.</p><p><b>RESULTS</b>mRNA expression levels of Notch1, Notch2, BCL-2 and NF-κB gene in CLL group were significantly higher than those in healthy control group (0.8556 ± 0.8726 vs 0.6731 ± 0.5334, P = 0.0182; 1.2273 ± 0.8207 vs 0.6577 ± 0.6424, P < 0.0001; 8.0960 ± 7.5661 vs 0.5969 ± 0.4976, P < 0.0001; 1.0966 ± 0.6925 vs 0.5373 ± 0.7180, P < 0.0001, respectively), but no significant difference was found between Notch3 and Notch4 gene (1.1914 ± 2.4219 vs 0.8713 ± 0.7937, P = 0.3427; 0.8174 ± 1.0869 vs 0.9752 ± 1.3446, P = 0.2402, respectively). Notch1 protein expression in L1210 cells were significantly decreased after treating with cytosine arabinoside of low and middle concentrations, but increased after treating with cytosine arabinoside of high concentration or prolonging time of cytosine arabinoside of middle con-centration. Notch1 protein expression in L1210 cells dereased after treating with dexamethasone, but did not be changed with the different concentrations and different times of dexmethason.</p><p><b>CONCLUSION</b>The transcription level of Notch gene in CLL patients significantly higher than that in normal controls. The Notch1 protein expression is down-regulated in process of inhibiting L1210 cell proliferation by Ara-C and dexmethason. Notch signaling pathway may mediated anti-apoptosis and drug resistance of CLL cells. Notch molecule possibly plays an important role in the anti-apoptosis and drug-resistance of CLL cells.</p>

Expression of DNA Methyltransferase Genes and Its Significance in AML Patients / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To detect the expression of DNA methyltransferases (DNMT) mRNA in the patients with acute myelogenous leukemia (AML) and to analyze the retationship between the mRNA expression of DNMT and cellular and moleculogenetic risk stratifieation in AML patients, and to evaluate the role of the DNMT mRNA expression in AML prognosis and clinical treatment.</p><p><b>METHODS</b>The mRNA expression of DNMT was detected by real-time PCR in 123 AML patients and 20 healthy people.</p><p><b>RESULTS</b>the mRNA expression levels of DNMT were lower in the healthy people and higher in AML patients; the mRNA expression levels of DNMT in the patients after the consolidation therapy were lower than that in the patients of initial diagnosis and replapse; The mRNA expression levels of DNMT did not correlate with age, sex and the clinical characteristics at initial diagnosis, such as white blood cell count, FAB classification and chromosomal karyotype in AML patients. In CR patients after standard treatment, the initial mRNA expression level of DNMT3b was higher. Based on cellular and moleculogenetic risk stratificantion, the DNMT expression level in the intermediate risk AML patients was higher.</p><p><b>CONCLUSION</b>The mRNA expression of DNMT may play an important role in AML pathogenesis and can serve as an index for evaluating AML prognosis and for instructing clinical treatment.</p>

Effect of Bortezomib on Proliferation, Apoptosis and SHP-2 Gene Expression of Lymphoma Cells / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate the effects of Bortezomib on proliferation, apoptosis and SHP-2 gene expression of lymphoma Jurkat cells and Raji cells.</p><p><b>METHODS</b>Methylthiazoly tetrazolium assay (MTT) was used to observe the proliferation of Jurkat cells and Raji cells treated with bortezomib in different doses. Cell apoptosis was detected by morphological examination and flow cytometry. The level of SHP-2 mRNA expression before and after the treatment with bortezomib was measured by RT-PCR.</p><p><b>RESULTS</b>Bortezomib could inhibit the proliferation of Jurkat and Raji cells and induce their apoptosis with time-and dose-dependent manner. After treatment with 5-100 nmol/L bortezomib, the expression of SHP-2 in Jurkat cells and Raji cells was upregulated.</p><p><b>CONCLUSION</b>Bortezomib can inhibit the proliferation and induc the apoptosis of Jurkat and Raji cells obviously, upregulate the expression of SHP-2 mRNA, suggesting that the SHP-2 may participate in regulation of bortezomib induced apoptosis of Jurkat cells and Raji cells.</p>

Research Progress on DKK1 Gene in Leukemia / 中国实验血液学杂志

A number of studies have demonstrated that the methylation of Dickkopf-1 (DKK1) gene promoter is related with the occurrence and development of many neoplastic diseases. By means of binding with corresponding receptors, DKK1 blocks the transduction pathway of Wnt/β-catenin/TCF and inhibits the proliferation and invasion of tumor cells, inducing apoptosis. Leukemia is a hyperplastic disease of hematopoietic stem cell malignant clone. Its pathogenesis has been confirmed to be closely related with the aberrant activation of Wnt signaling pathway. This pathway is associated with the self-renewal and proliferation of the hematopoietic stem cells, which can regulate growth, differentiation, migration of the cells, angiogenesis and embryonic development. Its expression is regulated by some suppressor genes like Dickkopf 1 (DKK1). Leukemia often accompanied by methylation modification of the DKK1 gene, so as to leads to silencing itself and activation of the Wnt signaling pathway, which cause the occurrence of leukemia. Some therapeutic methods on leukemia aiming at DKK1 gene have been reported, among which DKK1 gene was demethylated. The intensive study on the expression and function of DKK1 should be important for the early diagnosis, treatment and prognosis. This article reviews the current progress in this field.

Effect of angular pyranocoumarin isolated from peucedanum praeruptorum on the proliferation and apoptosis of U266 cells / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the effects of angular pyranocoumarin (±) -4'-O- acetyl-3'-Oangeloyl- cis- khellactone (APC) extracted from peucedanum praeruptoruon on the proliferation and apoptosis of U266 cells, and to explore its related mechanism.</p><p><b>METHODS</b>APC was extracted by petroleum ether technique, and its purity was tested by high performance liquid chromatography, and its chemical structure was identified by magnetic resonance spectroscopy. U266 cells were treated with APC in various concentrations (0, 10, 20, 30, 40 μg/ml）for different durations（24 and 48 h). The inhibitive effect of APC on cell growth was detected by CCK-8 method. After U266 cells were incubated with APC（0, 10, 20, 30, 40 μg/ml）for 24 h, the apoptosis of cells were observed by flow cytometry stained with Annexin Ⅴ/PI and Hochest33342; the expression levels of caspase-3, 8, ERK, p-ERK, AKT and p-AKT protein were assayed by Western blot; the expression of hTERT mRNA was measured by RT-PCR.</p><p><b>RESULTS</b>The purity of APC identified by magnetic resonance imaging was 98.8%. The proliferation of U266 cells was inhibited, and the apoptosis was induced in a time- and/or dose- dependent manner after treatment with APC. APC could upregulate the caspase- 8, 3 protein expression and downregulate the p- ERK, p-AKT protein expression along with the increase of APC dose. APC also could downregulate the hTERT mRNA expression.</p><p><b>CONCLUSION</b>Angular pyranocoumarin APC could inhibit the proliferation and induce the apoptosis of U266 cells. The probable mechanism might be achieved by upregulating caspase-8, 3 protein expression and downregulating p-ERK, P-AKT protein and the hTERT mRNA expression.</p>

A comprehensive analysis of breakpoint cluster region-abelson fusion oncogene splice variants in chronic myeloid leukemia and their correlation with disease biology.

BACKGROUND: BCR-ABL fusion oncogene is a hallmark of Chronic Myeloid Leukemia (CML). It results due to translocation between chromosome 22 and chromosome 9 [t (9; 22)(q34; q11)]. It gives rise to translation of a 210 KDa chimeric protein (p210), leading to enhanced tyrosine kinase activity and activation of leukemogenic pathways, ultimately causing onset of CML. In case of CML, the classic fusions are b2a2 or b3a2, fusing exon 13 (b2) or exon 14 (b3) of BCR, respectively, to exon 2 (a2) of ABL. The type of BCR-ABL transcripts are thought to be have different prognosis and hence useful in clinical decision-making. The frequencies of different fusion oncogenes associated with leukemia can vary in different ethnic groups and geographical regions due to interplay of genetic variation in different ethnic populations, diverse environmental factors and living style. Moreover, earlier relevant studies from our region were carried out in small subset of patients. Therefore, objective of this study was to find out frequencies of different BCR-ABL splice variants in larger subset of CML patients. METHODS: A nested reverse transcriptase polymerase chain reaction (RT-PCR) was established to detect BCRABL splice variants in 130 CML patients. Sensitivity of RT-PCR and ability to detect BCR-ABL fusion gene in least possible time was studied. RESULTS: BCR-ABL detection using our optimized RTPCR protocol could be completed in 8 hours, starting from RNA extraction to Gel electrophoresis. Sensitivity of RTPCR assay was of the order of 10−6. Out of 130 Pakistani patients, 83 (63.84%) expressed b3a2 while 47 (36.15%) expressed b2a2 transcript. CONCLUSION: Our RT-PCR was proved to be very quick to detect BCR-ABL fusion oncogene in CML patients within one working day. Because of its sensitivity, it can be used to monitor complete molecular response in CML. BCR-ABL RT-PCR and BCR-ABL splice variants frequency in our study differs from other ethnic groups. It shows that ethnic and geographical differences exist in BCR-ABL splice variant frequency, which may have a profound effect on disease biology as well as implications in prognosis and clinical management of BCR-ABL positive leukemias.

Effect of COX-2 inhibitor celecoxib on proliferation, apoptosis of HL-60 cells and its mechanism / 中国实验血液学杂志

This study was aimed to investigate the effect of COX-2 inhibitor celecoxib on proliferation, apoptosis of human acute myeloid leukemia cell line HL-60 and its mechanism. HL-60 cells were cultured with different concentrations of celecoxib for 24 h. Cell proliferation was analyzed by CCK-8 assay, cell apoptosis and cell cycle distribution were detected by flow cytometry. Cyclin D1, cyclin E1 and COX-2 mRNA expressions were determined by RT-PCR. The results showed that after the HL-60 cells were treated with different concentrations of celecoxib for 24 h, the cell growth was significantly inhibited in a dose-dependent manner(r = 0.955), IC50 was 63.037 µmol/L of celecoxib. Celecoxib could effectively induce apoptosis in HL-60 cells also in dose-dependent manner(r = 0.988), blocked the HL-60 cells in the G0/G1 phase. The expression of cyclin D1, cyclin E1 and COX-2 mRNA were downregulated. It is concluded that celecoxib can inhibit the proliferation of HL-60 cells in dose-dependent manner, celecoxib causes cell G0/G1 arrest and induces cell apoptosis possibly through down-regulation of the cyclin D1 and cyclin E1 expression, and down-regulation of COX-2 expression respectively.

Apoptosis-inducing effect of tetrandrine and imatinib on K562/G01 cells and its related mechanism / 中国实验血液学杂志

This study was purposed to explore the apoptosis-inducing effect of tetrandrine (Tet) and imatinib (IM) alone or both combined on K562/G01 cells and their mechanism. MTT assay was used to detect the inhibitory effect of drugs on cell growth, flow cytometry was used to detect the cell cycle and apoptosis rate. The expression of caspase-3/BCL-2 mRNA was determined by real time-PCR, and the expression of caspase-3/BCL-2 protein was assayed by Western blot. The results showed that after being treated by 1.0 µmol/L IM or 1.5 µmol/L Tet alone and combination of these two drugs for 48 h, the inhibitory rate was (22.74 ± 0.05)%, (20.34 ± 0.57)% and (44.28 ± 0.60)%, respectively, suggesting that inhibitory effect of two drug combination was more obvious. The arrest of cell cycle at G1/S phase could be observed after Tet treatment. Early apoptosis rate was (7.81 ± 0.16) %, (14.10 ± 0.28) % respectively after being treated by combination of 1.5 µmol/L and 3.0 µmol/L Tet with 1.0 µmol/L IM. After being treated with Tet alone, FQ-PCR and Western blot showed that the expressions of caspase-3 mRNA and caspase-3 protein were up-regulated, the expressions of BCL-2 mRNA and protein were down-regulated, the effect of both drug combination was more significant. It is concluded that IM or Tet alone can induce apoptosis of K562/G01. Combination of IM with Tet shows obvious synergistic effect, mechanism of which may associate with up-regulation of caspase-3 mRNA and protein expressions, and down-regulation of BCL-2 mRNA and protein expressions.

Bioinformatic analysis of chronic myeloid leukemia progression and preliminary experimental verification / 中国实验血液学杂志

This study was aimed to explore the progression mechanism of chronic myeloid leukemia, so as to provide the new molecular markers for evaluation of CML clinical outcome and selection of treatment. The microarray data of genes related with progression from different phase of chronic myeloid leukemia (CML) were collected from public data depository GEO (Gene expression datasets). SAM analysis, fold change filtering, cross comparison were used to analyze the data and identify different genes. Moreover, MeV and pSTIING sofewares were used to analyze the key differential genes and signal pathways. At last, Q-PCR were used to confirm the predicted key gene. The results indicated that after comparison, 9 genes were differentially expressed from AP to BC, and the integrin-mediated cell adhesion , focal adhesion, regulation of actin cytoskeleton were the principal pathways during CML progression. Network construction analysis found that AP-related genes or pathways may be the original signals; and MLLT4, WDR35 and EPHB4 were the key genes for CML progression. EPHB4 was confirmed by Q-PCR in CML BC patients and CP patients. It is concluded that MLLT4, WDR35, EPHB4, integrin-mediated cell adhesion, focal adhesion and regulation of actin cytoskeleton are the principal genes and pathways during CML progression.

Mutation and expression of PAX5 gene in adult acute lymphoblastic leukemia / 中国实验血液学杂志

PAX5 is an important transcription factor of paired-box(PAX) family. The aim of this study was to investigate the mutations and expression of PAX5 and its clinical significance in adult patients with acute lymphoblastic leukemia (ALL). Reverse transcription polymerase chain reaction (RT-PCR) and genomic PCR were performed to detect the deletions of PAX5 and point mutations of PAX5 exon 2-10 in 101 cases of adult ALL and were confirmed by cloning and sequencing. In addition, quantitative PCR (qPCR) was performed to evaluate the expression of PAX5. Furthermore, the correlations of mutations and expression of PAX5 with clinical parameters were analyzed, and the prognostic significance was evaluated as well. The results showed that PAX5 mutations were observed in 8 of 101 (7.9%) patients with B-ALL. A total of 9 types of mutations were detected, including 4 types of deletions, 4 types of point mutations and 1 insertion mutation; percentage of patients with age ≥ 50 years was higher in PAX5 mutation group than in wide-type group (62.5% vs 21.5%,P = 0.031) . The statistical differences were observed in B-cell subtype, initial platelet count and immunophenotypes between high and low expression of PAX5 (P < 0.05) . In addition, patients with high expression of PAX5 had higher first complete remission rate (86.7% vs 62.5%, P = 0.030) and 6-month overall survival rate (75.0% vs 50.0%, P = 0.034) compared with patients with low expression of PAX5. It is concluded that deletion/insertion/point mutations and aberrant expression of PAX5 can be observed in adult patients with B-ALL. Mutations and aberrant expression of PAX5 correlated with clinical parameters and have important clinical significance.

Mutation and expression of LEF1 in adult acute lymphocytic leukemia and their clinical significance / 中国实验血液学杂志

Lymphoid enhancer factor 1 (LEF1) is a key transcription factor in Wingless-type (Wnt) pathway. The present study was aimed to explore the genetic mutation and expression of LEF1, and their clinical significance in adult patients with acute lymphocytic leukemia (ALL). Genomic DNA was amplified and sequenced to detect the mutation of LEF1 in 131 newly diagnosed adult patients with ALL. Quantitative PCR (qPCR) was performed to detect the expression of LEF1. Moreover, the correlations between mutations and expression of LEF1 with clinical characteristics were analyzed. The results showed that the frequency of LEF1 mutation in adult ALL was 3.1% (4/131) and all of them were point mutations located in exon 2 and 3; the median white blood cell count and median percentage of blasts at diagnosis were significantly higher in LEF1 high expression group than in low expression group (70.6 × 10⁹/L vs 26.2 × 10⁹/L)(P = 0.010); (81.0% vs 57.0%) (P = 0.014); in addition, the percentage of patients with Philadelphia chromosome positive and patients in high-risk group significantly increased in LEF1 high expression group compared with that in low expression group (66.7% vs 36.5%) (P = 0.038); (79.2% vs 56.2%) (P = 0.044). It is concluded that high expression of LEF1 may play an important role on development of adult ALL.

Effects of arsenic trioxide on the methylation of TMS1 gene in K562 cells / 中华血液学杂志

<p><b>OBJECTIVE</b>To detect the methylation status of TMS1 gene and its demethylation by arsenic trioxide (As₂O₃) in K562 cells.</p><p><b>METHODS</b>K562 cells were treated with different concentrations of As₂O₃ for 48 hours. Methylation-specific PCR (MSP) was used to determine the methylation status of TMS1. RT-PCR and Western blot were used to detect the levels of TMS1 mRNA and protein. TMS1 associated apoptosis proteins Bcl-2/Bax were also analyzed by Western blot. Apoptosis were evaluated by flow cytometry using Annexin V/propium iodide (PI) double staining.</p><p><b>RESULTS</b>TMS1 gene was completely methylated in K562 cells and the levels of TMS1 mRNA and protein were low (0.01±0.01, 0.09±0.02), which could be reversed (mRNA: 0.72±0.04; protein: 1.30±0.06; P<0.01) by 2 μmol/L As2O3 via overt demethylation of TMS1 gene. Apoptosis in experiment group (12.24±1.06) was significantly higher than that in control group (2.05±0.16, P<0.05). In experiment group, the down-expression of antiapoptotic protein Bcl-2 and up-expression of pro-apoptotic protein Bax led to an obvious decline ratio of Bcl-2/Bax (0.56±0.12), as compared to the control group (1.94±0.14, P<0.01).</p><p><b>CONCLUSION</b>As₂O₃ could up-regulate TMS1 gene expression by reversing its hypermethylation and induced apoptosis by down-regulation of Bcl-2/Bax ratio in K562 cells.</p>

Expression of musashi-2 gene in leukemia stem cells from acute myeloid leukemia patients / 中国实验血液学杂志

This study was aimed to detect the expression of Musashi-2 (Msi2) in acute myeloid leukemia (AML) and investigate the relationship between Msi2 and other clinical parameters, especially CD34. A total RNA was extracted from bone marrow of newly diagnosed AML patietns. The Msi2 mRNA expression in newly diagnosed AML patients was detected with real-time fluorescence quantitative RT-PCR. The expression level of CD34 in above-menthioned patients was detected by flow cytometry (FCM). The relationship between the expression of Msi2 mRNA and clinical outcome in AML patients was analysed. The results showed that (1)the expression of Msi2 mRNA in newly diagnosed AML patients was much higher than that in healthy volunteers (P < 0.05) , especially in M1, M4 and M5 patients; (2)the expression level of Msi2 did not correlate with age, sex, white blood cell count of peripheral blood, AML1/ETO and PML/RARa fusion gene (P > 0.05); (3) Msi2 expression level in patients with CD34(+) cells was significantly higher than that in patients with CD34(-) cells (P < 0.05). It is concluded that the Msi2 mRNA expresses in leukamia stem cells, the high expression of Msi2 mRNA has been found in newly diagnosed AML patients, especially in M1, M4 and M5 patients, the high expression also has been observed in patients with CD34(+).