OBJECTIVETo observe the effect of leukemic cells on blood-brain barrier (BBB) in mice with central nervous system leukemia (CNSL) by establishing mice CNSL model and an in vitro BBB model and explore the mechanism of leukemic cell infiltrating central nervous system (CNS).

METHODSAfter splenectomy, cytoxan intraperitoneal injection, and sublethal irradiation, 10 BALB/c nu/nu mice were transplanted intravenously with 1.2 × 10(7) of SHI-1 human monocytic leukemic cells. Mice were monitored for survival and clinical manifestation of nerve palsy. The leukemic cells engrafted were examined by RT-PCR, histopathology and bone marrow (BM) smears. Immunofluorescence analysis with laser scanning fluorescence confocal microscopy was used to determine the expression of fibrinogen and tight-junction protein ZO-1. An in vitro BBB model composed of human brain microvascular endothelial cells (BMVECs) was developed on a Matrigel-based insert. Different leukemic cell lines were seeded onto the upper compartment of transwell insert. After incubated for 24 h with BMVECs, cells that had migrated into the lower compartment were counted and analyzed.

RESULTS(1) Paralysis with or without sight loss was developed in half the mice 30-35 d after innoculated with SHI-1 cells. Leukemic cells infiltrates were observed in BM and in different part of brain tissues including brain parenchyma. The transcriptions of human MLL/AF6 fusion gene were also detected in BM and brain tissues in paralysis mice. The fibrinogen expression and ZO-1 disruption were detected in the infiltrated tissue. (2) After 24 h incubation with leukemic cells, the BMVECs sheets were disrupted and grew singly and ZO-1 expression was down-regulated markedly. SHI-1 cells showed more injurious to BMVECs and higher invasive rate \[(40.33 ± 1.53)% vs (11.83 ± 1.44)%, P < 0.05\] than HL-60 cells did.

CONCLUSIONOne of the mechanisms of leukemic cells infiltrates CNS in CNSL is injure to the BBB.

Animals ; Blood-Brain Barrier ; physiology ; Central Nervous System ; pathology ; Central Nervous System Neoplasms ; pathology ; HL-60 Cells ; Humans ; Leukemia ; pathology ; Mice ; Mice, Inbred BALB C ; Mice, Nude

OBJECTIVETo study the relationship between pretransplantation host thymic recent output function and prognosis in HLA-matched sibling bone marrow transplantation (MSD-BMT) and determine whether pretransplantation host thymic recent output function can act as a marker for predication of prognosis after HSCT.

METHODST-cell receptor excision circle (TREC) in DNA of pretransplantation peripheral blood mononuclear cells from 64 patients underwent MSD-BMT was detected by real-time quantitative PCR. The content of TREC in 70 normal donors was detected as well. All clinical data of patients after HSCT were collected and studied. Survival rates of patients after HSCT were estimated with Log-rank test. Univariate and multivariate analysis of prognostic factors were carried out by COX's proportional hazard regression model.

RESULTSThe mean value of TREC in normal donors was (3351 +/- 3711) copies/10(5) cells. There was an inverse correlation between TREC and age in the donor groups. Before transplantation, all patients were detected TREC, with a mean TREC number of (180 +/-332) copies/10(5) cells being significantly lower than that of normal donors. The results of univariate analysis showed that the counts of pre-HSCT TREC were closely, correlated with long term survival and chronic graft versus host disease (cGVHD) (P < 0.05) and with CMV infection (P = 0.084) but not with acute graft versus host disease (aGVHD). The results of multivariate analysis showed the same thing as that of univariate analysis.

CONCLUSIONPretransplantation host thymic recent output function is closely correlated with prognosis in MSD-BMT and can be a factor for predicting the outcome of HSCT.

Adolescent ; Adult ; Child ; Female ; Hematopoietic Stem Cell Transplantation ; methods ; Humans ; Male ; Middle Aged ; Prognosis ; Receptors, Antigen, T-Cell ; genetics ; Retrospective Studies ; Siblings ; Thymus Gland ; immunology ; Transplantation, Homologous ; immunology ; methods

The identification of genes inducing resistance to anticancer chemotherapeutic agents and their introduction into hematopoietic cells represents a promising approach to overcome bone marrow toxicity, the limiting factor for most high-dose chemotherapy regimens. Because resistance to cyclophosphamide has been correlated with increased levels of expression of the aldehyde-dehydrogenase (ALDH1) gene in tumor cells lines in vitro, this study tested whether ALDH1 overexpression could directly induce cyclophosphamide resistance. Results showed that a retroviral vector was used to transduce full-length human ALDH1 cDNA into human hematopoietic cell line K562 that was then tested for resistance to 4-hydroxycyclophosphamide (4-HC), an active analogue of cyclophosphamide. Overexpression of the ALDH1 gene resulted in a significant increases in cyclophosphamide resistance in transduced K562 cells (50% inhibition concentration, IC50 = 10 micro mol/L). These findings indicate that ALDH1 overexpression is sufficient to induce cyclophosphamide resistance in vitro and provide a basis for testing the efficacy of ALDH1 gene transduction to protect bone marrow cells from high-dose cyclophosphamide in vivo.
Aldehyde Dehydrogenase ; genetics ; Antineoplastic Agents, Alkylating ; pharmacology ; Cell Division ; drug effects ; Cell Survival ; drug effects ; Cyclophosphamide ; analogs & derivatives ; pharmacology ; Dose-Response Relationship, Drug ; Drug Resistance, Neoplasm ; Gene Expression Regulation, Enzymologic ; Genetic Vectors ; genetics ; Humans ; Inhibitory Concentration 50 ; K562 Cells ; drug effects ; enzymology ; metabolism ; Retroviridae ; genetics ; Transfection

OBJECTIVETo investigate Wilms' tumor gene (WT1) expression levels in bone marrow (BM) of acute leukemia patients (ALs).

METHODSA real-time quantitative reverse transcriptase polymerase chain reaction (RQ-RT-PCR) method was established for detecting WT1 and internal reference GAPDH expression levels in BM of 108 ALs and 23 non-leukemia controls by Light Cycler.

RESULTSThe median expression levels of WT1 in 70 newly diagnosed ALs and 11 relapsed ALs were statistically higher than those in 23 ALs in complete remission (CR) and 23 non-leukemic controls (75.10 and 89.56 vs 2.07 and 1.51 respectively). No statistic differences was found between the CR group and control group, nor between the newly diagnosed group and relapsed group. Of the 70 newly diagnosed ALs, median WT1 expression level of acute granulocytic leukemias was significantly higher than that of acute monocytic leukemias (M(5)), but there was no statistic differences among the M(1), M(2), M(3) and ALL subtypes. Furthermore the WT1 levels were not correlated to peripheral WBC counts, BM blast percentage and multidrug resistant gene (mdr1) expression at presentation, but correlated to chromosome karyotypes. Dynamic analysis of WT1 levels of 2 patients on treatment showed that WT1 expression levels predicted relapse.

CONCLUSIONWT1 expression levels in ALs were strikingly higher than that in non-leukemias. WT1 can be a marker for detecting MRD and evaluating therapy efficacy in leukemias.

Acute Disease ; Adolescent ; Adult ; Aged ; Bone Marrow Cells ; metabolism ; Child ; Female ; Gene Expression Regulation, Leukemic ; Humans ; K562 Cells ; Leukemia ; blood ; genetics ; Leukemia, Monocytic, Acute ; blood ; genetics ; Leukemia, Myeloid ; blood ; genetics ; Male ; Middle Aged ; Reverse Transcriptase Polymerase Chain Reaction ; methods ; WT1 Proteins ; genetics ; Young Adult

The objective of this study was to identify the frequency and types of JAK2V617F mutation in chinese patients with essential thrombocythemia (ET), to quantitatively detect the level of mutation transcripts and to investigate its clinical significance. The frequency and types of JAK2V617F mutation were detected by amplification-refractory mutation sequencing polymerase chain reaction (ARMS-PCR), the transcript level of JAK2V617F mutation was determined by using capillary electrophoresis. The results indicated that the JAK2V617F mutation was detected in 59 out of 98 patient with ET, 18 of whom were homozygous mutation. The mean age of patients with homozygous and heterozygous mutation was higher than that of patients with wild type mutation (p < 0.05). The quantitative assay using capillary electrophoresis showed that the transcript level of JAK2V617F mutation in patients with homozygous mutation was (89.9 +/- 6.7)%, which was higher than that in patients with heterozygous mutation (57.1 +/- 6.7)% (p < 0.05); the transcript level of JAK2V617F mutation in patients with age < 60 years was (62.3 +/- 16.5)%, which was lower than that in patients with age > 60 years (72.4% +/- 15.8)% (p < 0.05). The rate of thrombotic complications in patients with JAK2V617F-positive was higher than that in patients with JAK2V617F-negative in which the rate of thrombotic complication in patients with homozygous mutation was higher than that in patients with heterozygous mutation (p < 0.05). Compared with patients without thrombotic events, there were higher level of transcripts of JAK2V617F mutation in patients with thrombotic events. It is concluded that the JAK2V617F positive and negative patients with ET display the different clinical features, therefore, the analysis of mutation types and detection of transcript levels not only helps to identify the disease status and progression, but also guides the treatment of ET patients.
Adolescent ; Adult ; Aged ; Aged, 80 and over ; Female ; Genotype ; Humans ; Janus Kinase 2 ; genetics ; Male ; Middle Aged ; Mutation ; Thrombocythemia, Essential ; genetics ; pathology ; Young Adult

OBJECTIVETo investigate the frequency and mutational status of JAK2 mutation in Chinese patients with chronic myeloproliferative neoplasms (MPN) and study the relative quantitation and clinical implications of mutated JAK2 transcript.

METHODSJAK2 mutation and the mutational status were screened with amplification-refractory mutation sequencing polymerase chain reaction (ARMS-PCR), the relative quantity of mutated JAK2 mRNA by using capillary electrophoresis.

RESULTSJAK2V617F mutation was detected in 95 of 135 MPN patients, including 37 (97.4%) of 38 polycythemia vera (PV), 56 (59.6%) of 94 essential thrombocythemia (ET) and 2 of 3 idiopathic myelofibrosis (IMF) patients; the difference between the mutations in PV and ET was significant (P<0.05). Of 95 JAK2V617F patients examined, 18/38 PV patients (47.3%) and 17/94 (18.1%) ET patients and 1 IMF patient were homozygotes, and ET patients showed lower prevalence of homozygote (P<0.05). In 95 MPN patients, the mutated mRNA ratio was higher in homozygote than in heterozygote patients. PV heterozygote patients showed higher levels of mutated JAK2 mRNA than ET heterozygote patients (P<0.05). The levels of JAK2V617F mRNA in patients over 60 years of age were significantly higher than that in those less than 60 years of age (P<0.001). Higher leukocyte counts were observed in PV and ET patients with higher levels of mutated JAK2 mRNA (P<0.05). The presence of JAK2V617F was found to be significantly associated with higher hemoglobin level in ET patients. Cytogenetic analysis was performed in 101 of the 135 patients, the association between abnormal karyotype and JAK2V617F was not found.

CONCLUSIONThe ARMS-PCR technique can be used to detect the frequency and mutational status of JAK2V617F mutation, and along with capillary electrophoresis, the estimation of minimal residual disease becomes possible.

Adolescent ; Adult ; Aged ; Aged, 80 and over ; DNA Mutational Analysis ; Electrophoresis, Capillary ; Female ; Humans ; Janus Kinase 2 ; genetics ; Male ; Middle Aged ; Mutation ; Myeloproliferative Disorders ; genetics ; Polymerase Chain Reaction ; methods ; RNA, Messenger ; genetics ; Young Adult

OBJECTIVETo evaluate the prevalence of Fms-Like tyrosine kinase 3 (FLT3) mutations including internal tandem duplication (ITD) of juxtamembrane region and point mutation in the second tyrosine kinase domain (TKD) in acute promyelocytic leukemia (APL) and its clinical significance.

METHODSBone marrow mononuclear cells from 160 newly diagnosed APL patients were analyzed. Polymerase chain reaction (PCR) was used to detect FLT3-ITD mutations, FLT3-ITD positive samples were further analyzed for the ITD allelic ratio (ITD-AR, mutant-wild type ratio). The FLT3-TKD mutation was analyzed by PCR amplification of exon 20 followed by EcoR V digestion and sequencing.

RESULTSOut of 160 patients, 30 (18.75%) patients were FLT3-ITD positive, 17 (10.62%) were FLT3-TKD positive, 2 had both of mutations. The initial WBC count and the ratio of short type PML-RAR alpha isoforms in FLT3-ITD positive and FLT3-TKD positive patients were all higher than that in patients with wild-type FLT3 (FLT3-wt) (P < 0.05). For FLT3-ITD positive patients, the incidences of retinoic acid syndrome (RAS) and disseminated intravascular coagulation (DIC) were 41.7% and 65.4%, respectively, being higher than that of FLT3-wt patients, while their complete remission (CR) rate was lower (69.2% vs 90.3%, P < 0.05). For FLT3-TKD positive patients, the incidence of RAS, DIC and CR rate were not significantly different from that of FLT3-wt patients (P > 0.05). FLT3-ITD positive patients had a shorter overall survival (OS) (P < 0.05), but not disease-free survival (DFS) (P > 0.05) as compared with FLT3-wt patients. There was no significant difference in either OS or DFS between FLT3-TKD positive and FLT3-wt patients. The ITD-AR of 30 FLT3-ITD positive patients varied from 0.11 to 6.55 with a median of 1.0. The initial WBC count, incidence of RAS and DIC, CR rate were not significantly different between the patients with ITD-AR greater than 1.0 and lower than 1.0 (P > 0.05).

CONCLUSIONSFLT3 mutations (FLT3-ITD or FLT3-TKD) are frequently identified in patients with newly diagnosed APL, both mutations are associated with higher initial WBC and short type PML-RAR alpha isoforms. FLT3-ITD mutation is more frequent than FLT3-TKD mutation, and predicts a poorer prognosis, whereas FLT3-TKD mutation does not show the same unfavorable prognostic effect on APL patients.

Adolescent ; Adult ; Aged ; Child ; Female ; Humans ; Leukemia, Promyelocytic, Acute ; diagnosis ; genetics ; Male ; Middle Aged ; Point Mutation ; Prognosis ; Tandem Repeat Sequences ; Young Adult ; fms-Like Tyrosine Kinase 3 ; genetics

OBJECTIVETo evaluate the prevalence of nucleophosmin (NPM1) gene exon 12 mutations in adults with acute myeloid leukemia (AML) and its clinical characteristics.

METHODSGenomic DNAs from 101 AML adults were screened by PCR and sequencing or capillary electrophoresis (CE) for NPMI mutations.

RESULTSNPM1 exon 12 mutations were present in 31.7% of the overall cohort, including 1/1 (100%) of M0, 9/17(52.9%) of M1 , 7/25 (28.0%) of M2, 0/23(0%) of M3, 2/7 (28.6%) of M4 and 13/25 (52.0% ) of M5. NPM1 gene mutations were more prevalent in patients with normal karyotype (27/59, 45.8%) compared with that in those with karyotypic abnormalities (5/42, 11.9% ) (P < 0.001). NPM1 mutant cases were significantly associated with old age (P < 0.05), high peripheral white cell count (P < 0.05) and low expression of CD34 (P < 0.05) and CD17 (P<0.05). Sequence analysis of these NPM1 mutant cases revealed 5 known mutations (type A, B, D, N(M), and P(M)) and 1 novel variant (named as type S).

CONCLUSIONSNPM1 exon 12 mutations occur with a considerable percentage in AML patients with normal karyotype, M1/M5 subtype and older age, and are associated with higher peripheral white cell count and lower expression of CD34 and CD117.

Adolescent ; Adult ; Aged ; DNA Mutational Analysis ; Exons ; Female ; Humans ; Leukemia, Myeloid, Acute ; genetics ; Male ; Middle Aged ; Mutation ; Nuclear Proteins ; genetics

This study was aimed to explore the contribution of autophagy associated gene Beclin1 in the prognosis of myelodysplastic syndrome (MDS) by detecting the expression level of Beclin1 in bone marrow mononuclear cells (BMNC) from 40 MDS patients, 14 non-malignant anemia patients and 25 AML patients. The expression of Beclin1 mRNA was detected by real-time quantitative polymerase chain reaction (qRT-PCR). At the same time, the Western blot was used to analyze the expression of Beclin1 proteins. The results showed that the expression of Beclin1 in low risk MDS patients and non-malignant anemia patients was both significantly higher than that in acute myeloid leukemia patients (P < 0.01). And more interestingly, the Beclin1 mRNA expression in MDS group was negatively correlated with World Health Organization classification-based prognostic system (WPSS) score (r = -0.495). It is concluded that the expression of Beclin1 in the patients with MDS is higher than that in AML patients, and negatively correlated with WPSS scores. Beclin1 is a potential biomarker for predicting prognosis of the patients with MDS.
Anemia ; metabolism ; Apoptosis Regulatory Proteins ; genetics ; metabolism ; Autophagy ; Beclin-1 ; Biomarkers, Tumor ; metabolism ; Bone Marrow Cells ; metabolism ; Humans ; Leukemia, Myeloid, Acute ; metabolism ; Membrane Proteins ; genetics ; metabolism ; Myelodysplastic Syndromes ; metabolism ; pathology ; Prognosis

OBJECTIVETo explore the role of bromodomain and extra terminal (BET) bromodomain in hematopoietic differentiation from human enbryonic stem cells (hESC).

METHODSThe effect of BET hematopoietic inhibitor I-BET151 on hematopoietic differentiation from hESC was detected by using a monolayer hematopoietic defferentiation model, immunofluorescence, flow cytometry and real-time PCR; moreover the role of I-BET151 in process of hematopoietic differentiation was explored by adding I-BET151 in different differentiation stages.

RESULTSThe analysis results of immunofluorescence, flow cytometry and real-time PCR showed that I-BET 151 significantly inhibited the generation of CD43 positive hematopoietic stem and progenitor cells (HSPCs). It was found that the addition of I-BET 151 in different stages, including APLNR lateral plate mesoderm production, CD34CD31 hemogenic endothelium (HEP) generation and endothelial-to-hematopoietic transition, significantly suppressed the generation of CD43 positive hematopoietic progenitor cells.

CONCLUSIONI-BET 151 inhibites hematopoietic differentiation from hESCs at several stages, suggesting that the BET bromodomain plays important roles in multiple stages of hematopoietic differentiation from hESCs.

Apelin Receptors ; Cell Differentiation ; Flow Cytometry ; Hemangioblasts ; Hematopoietic Stem Cells ; Human Embryonic Stem Cells ; Humans