Objective To investigate the point mutations within the adenosine triphosphate-binding region of BCR/ABL in patients with chronic myeloid leukemia who develop imatinib resistance.Methods We collected a total of 17 bone marrow samples obtained from 11 patients who showed hematology resistance (n = 7)or cytogenetic refractoriness(n = 4).A long semi-nest PCR method was used to amplify the ABL kinase domain of the BCR/ABL allele.After two rounds of PCR reactions,we got a fragment of 863 bases The PCR products were purified and followed by sequencing.Results In total,we find three point mutations presented in all patients tested G250E,E255K and T315I.The mutation rate of hematology resistance is 4/ 7,and 95% confidence interval was 8%-90%,while mutation rate of cytogenetic refractoriness 1/4,95% confidence interval 1%-81%.For those patients whose samples were available,no single mutation were determined before imatinib resistance emerged.Conclusions There is high frequency of point mutations clustered within the adenosine triphosphate-binding region of BCR/ABL in patients with chronic myeloid leukemia.It's good for patients to switch to another therapeutic strategy when the mutations are detected.

OBJECTIVETo study the potential effects of exogenous WT1 gene isoform on the differentiation of leukemia cell line NB4 and its possible molecular mechanisms.

METHODSThe recombinant eukaryotic expression vector (pCB6 + /WTA) containing full-length human WT1 isoform (WTA: -17AA/ -KTS) cDNA and the blank pCB6 + vector were transfected into leukemia cell line NB4 by electroporation. The WTA mRNA and protein in cells were detected by RT-PCR and Western blot. Cell morphology, NBT reduction and CD11b antigen expression in NB4 cells were assayed to evaluate cell differentiation. Expression of PML/RARalpha, p21 and c-myc genes was determined by semi-quantitative RT-PCR after transfection.

RESULTSCompared with NB4/WTA cells, NB4 and NB4/CMV (NB4 cells transfected with pCB6 + vector) cells had higher morphological differentiation rates and higher CD11b expression levels after exposure to ATRA for 48 hours. The percentage of NBT reduction in NB4/WTA cells was lower than that in control groups. The difference in NBT reduction rate between NB4/WTA and control cells was gradually increased after treated with ATRA for three days. The expression levels of PML/RARalpha, p21 and c-myc genes in NB4/WTA cells were notably increased.

CONCLUSIONOverexpression of exogenous WTA gene could partially inhibit the differentiation of NB4 cells by up-regulating the expression of PML/RARalpha, p21 and c-myc genes.

Cell Cycle ; Cell Differentiation ; genetics ; Cell Line, Tumor ; Cyclin-Dependent Kinase Inhibitor p21 ; metabolism ; Gene Expression Regulation, Neoplastic ; Genetic Vectors ; Humans ; Leukemia, Promyelocytic, Acute ; genetics ; metabolism ; pathology ; Proto-Oncogene Proteins c-myc ; metabolism ; RNA, Messenger ; genetics ; Transfection ; WT1 Proteins ; genetics ; metabolism

OBJECTIVETo study the DNA methylation status of WT1 gene promoter region in hematologic malignancy cell lines and its correlation with WT1 gene expression.

METHODS1. RT-PCR and methylation-specific PCR were performed for detecting WT1 gene expression and DNA methylation status in its promoter region in 8226, HL-60, Jurkat, K562, KG-1, NB4, Raji, SHI-1, U266 and U937 cell lines. 2. Treatment of U937 cells with 5-aza-CdR, a demethylation inducing agent and the changes in WT1 gene expression level and its promoter region methylation status were determined.

RESULTS1. HL-60, K562, KG-1, NB4 and SHI-1 cells showed higher levels while 8226, Jurkat, Raji, U266 and U937 cells showed extremely low levels of WT1 expression. DNA hypermethylation in WT1 gene promoter region was identified in 8226, Jurkat, Raji, U266 and U937 cells. 2. The WT1 gene expression in U937 was enhanced after treatment with 5-aza-CdR accompanied with the decrease of methylated and the increase of unmethylated levels in its promoter region.

CONCLUSIONModulation of the DNA methylation status in WT1 promoter region is one of the epigenetic mechanisms for regulating its expression.

Cell Line, Tumor ; DNA Methylation ; Gene Expression Regulation, Neoplastic ; Hematologic Neoplasms ; genetics ; Humans ; Promoter Regions, Genetic ; genetics ; WT1 Proteins ; genetics

OBJECTIVETo investigate retinoblastoma (Rb) associated protein 46 (RbAp46) gene expression levels in bone marrow (BM) cells of leukemia patients.

METHODSReal-time quantitative reverse polymerase chain reaction (QRT-PCR) method was used for detecting RbAp46 expression levels in BM cells of 140 patients with acute leukemia (AL), 13 with chronic myelogenous leukemia in chronic phase (CML-CP), 7 with CML in blast crisis (CML-BC) and 32 with non-leukemic disorders.

RESULTSThe M-Estimators of RbAp46 were higher in 98 newly diagnosed ALs and 5 relapsed ALs than in 28 ALs in complete remission (CR) and 32 non-leukemic controls (178.23 and 213.65 vs 85.89 and 88.08, respectively). No statistic difference was found between the CR group and control group, or between the newly diagnosed group and relapsed group. The M-Estimators of RbAp46 in patients with CML-CP was 58.27, similar to that in control, but much lower than that in CML-BC (173.24). Among 98 newly diagnosed ALs, the M-Estimators of RbAp46 in M(3) and M(4) were the lowest in all of the subtypes. Furthermore, the RbAp46 expression levels were not correlated with the expression of the fusion genes of bcr/abl, PML-RARalpha, and multidrug resistant gene (mdr1), but were positively correlated with Wilms' tumor gene (WT1) expression levels and negatively with AML1/ETO fusion gene expression.

CONCLUSIONRbAp46 expression levels in ALs and CML-BC were strikingly higher than that in non-leukemias and CML-CP, and might participate in leukemogenesis.

Adolescent ; Adult ; Aged ; Carrier Proteins ; genetics ; metabolism ; Child ; Female ; Gene Expression ; Humans ; Leukemia ; genetics ; metabolism ; Male ; Middle Aged ; Nuclear Proteins ; genetics ; metabolism ; Retinoblastoma-Binding Protein 7 ; Reverse Transcriptase Polymerase Chain Reaction ; methods

Chronic myeloid leukemia (CML) is a malignant clonal disease derived from hematopoietic stem cells. CML stem cells were thought to be the root which could lead disease development and ultimately rapid change. However, a stable animal model for studying the characteristics of CML stem cells is currently lacking. This study was aimed to establish a transplanted human CML nude-mice model to further explore the biological behavior of CML stem cells in vivo, and to enrich CML stem cells in nude mice by series transplantation. The 4 - 6 weeks old BALB/c nude mice pretreated by splenectomy (S), cytoxan intraperitoneal injection (C) and sublethal irradiation (I) were transplanted intravenously with (5 - 7) × 10(7) of bone marrow mononuclear cells from CML patients in chronic phase. Alternatively, 4 - 6 weeks old BALB/c nude mice pretreated by lethal irradiation were transplanted intravenously with 5 × 10(6) homologous bone marrow cells of BALB/c nude mice together with (5 - 7) × 10(7) of bone marrow mononuclear cells from CML patients in chronic phase simultaneously. The leukemic cells engrafted and infiltrated in organs and bone marrow of the mice were tracked by reverse transcription-polymerase chain reaction (RT-PCR), plastic-embedded biopsy and flow cytometry. The results of these two methods were compared. The results showed that human CML cells engrafted and infiltrating into the bone marrow of two nude mice pretreated with SCI could be detected. In spite of the low successful rate, results suggested the feasibility of this method by using BALB/c nude mice as a human CML animal model. In contrast, in nude mice pretreated by the lethal dose irradiation, CML cells in the bone marrow could not be found. It is concluded that human bone marrow CML cells can results in leukemia in nude mice pretreated by SCI. Thus this study provides a new strategy for establishment of CML animal models which deserves further elaboration.
Animals ; Disease Models, Animal ; Humans ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; Male ; Mice ; Mice, Nude ; Mice, SCID ; Neoplasm Transplantation ; Neoplastic Stem Cells ; Transplantation, Heterologous

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 investigate the effects of tributyrin (TB), a histone deacetylase inhibitor, on the growth, differentiation and apoptosis of SHI-1 leukemia cells and explore its possible mechanism.

METHODCell proliferation and viability were determined by cell counting, trypan blue dye exclusion. Cell morphological analysis, Annexin binding, DNA electrophoresis, expression of CD11b and CD14, NBT reduction were performed to evaluate differentiation and apoptosis of SHI-1 cells. The level of acetylated histone H3 was detected by Western blot and p21(WAF1/CIP1) expression by semi-quantitative RT-PCR.

RESULTSTB inhibited the proliferation and viability of SHI-1 cells in a time-dose dependent manner. The morphology of SHI-1 cells cultured in the presence of 0.1 mmol/L TB for 72 hs was more mature with higher NBT positivity and up-regulated expressions of CD11b and CD14 than that of control group. Exposed to 0.5 - 1.0 mmol/L TB for 48 hs, SHI-1 cells exhibited the morphological hallmarks of apoptosis, the increasing of Annexin binding and the DNA ladder on gel electrophoresis. The level of acetylated histone H3 and p21(WAF1) mRNA extracted from SHI-1 cells were increased by the treatment of TB.

CONCLUSIONTB can inhibit proliferation, induce differentiation and apoptosis of SHI-1 cells. The mechanism may associate with its up-regulation of acetylated histone and the expression of p21(WAF1).

Acetylation ; drug effects ; Apoptosis ; drug effects ; Blotting, Western ; Cell Differentiation ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cyclin-Dependent Kinase Inhibitor p21 ; genetics ; Enzyme Inhibitors ; pharmacology ; Gene Expression ; drug effects ; Histone Deacetylase Inhibitors ; Histone Deacetylases ; metabolism ; Histones ; metabolism ; Humans ; Leukemia, Monocytic, Acute ; genetics ; metabolism ; pathology ; Reverse Transcriptase Polymerase Chain Reaction ; Triglycerides ; pharmacology

OBJECTIVETo establish a model of human monocytic leukemia with CNS infiltration in BALB/c nude mice.

METHODSBALB/c nu/nu mice pre-treated by splenectomy, cytoxan intraperitoneal injection, and sublethal irradiation (SCI), were transplanted intravenously with 1 x 10(7) of human monocytic leukemic SHI-1 cells. The leukemic cells engrafted in the mice were detected by RT-PCR, histopathological examination, immunohistochemistry and FCM.

RESULTSThe survival time of SCI-nu/nu mice was 33-46 d. Paraplegia occurred in some of the mice. 5 weeks after transplantation, SHI-1 cells engrafted in SCI-nu/nu mice, multi-organs were involved and green solid neoplasms were formed in some organs. Histopathological examination found that SHI-1 cells infiltrated in liver, lung, kidney and testis of the mice and vertebral and skull bone marrow was replaced by leukemic cells. Leukemic cell penetrated through the surface of vertebrae, formed neoplasm, and entered the subdural space, but seldom involved the spinal parenchyma. In brain leukemia cells were filled in the subdural space and pia-arachnoid, covered the surface of cerebrum, cerebellum, spread along the virchow-robin space on the surface of pia mater, and eventually invaded the brain parenchyma.

CONCLUSIONSHI-1 cells could engrafted in the SCI-nu/nu mice, form an efficient and reproducible experimental model of CNSL and systematic leukemia. This model may be useful for studying the pathogenesis of CNSL.

Adult ; Animals ; Cell Line, Tumor ; Central Nervous System Neoplasms ; Humans ; Leukemia, Experimental ; pathology ; Leukemia, Monocytic, Acute ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Rats ; Xenograft Model Antitumor Assays ; methods

OBJECTIVETo investigate the prevalence of JAK2V617F and MPLW515L/K mutation in patients with slightly elevated platelets (BPC) or hemoglobin (Hb) not meeting the criteria of polycythemia vera (PV) or essential thrombocythemia (ET).

METHODSGenomic DNA from bone marrow or blood mononuclear cells was screened with allele specific polymerase chain reaction (AS-PCR) for JAK2V617F and MPLW515L/K mutation. The history of thrombosis was assessed retrospectively by patients files.

RESULTSOf 30 patients, 14 (46.7%) were positive for the JAK2V617F mutation, none of them had the MPLW515L/ K. Five of these 14 patients had a history of thrombosis. Follow-up results were available in 22 patients. Among them, 12 patients with JAK2V617F mutation turned out to be MPD in 6-24 months; only 2 out of 10 patients without this mutation evolved to MPD.

CONCLUSIONJAK2V617F mutation could be one of the diagnosis criteria of early MPD. No MPLW515L/K expression was found in early MPD.

Adolescent ; Adult ; Aged ; Aged, 80 and over ; Early Diagnosis ; Female ; Follow-Up Studies ; Humans ; Janus Kinase 2 ; genetics ; metabolism ; Male ; Middle Aged ; Mutation ; Myeloproliferative Disorders ; diagnosis ; genetics ; metabolism ; Receptors, Thrombopoietin ; genetics ; metabolism ; Young Adult

OBJECTIVETo investigate the influence of tissue inhibitor of metalloproteinase 2 (TIMP-2) on the infiltrative patterns of human monocytic leukemic cell line SHI-1 in nude mice.

METHODS1) 1 x 10(7) TIMP-2 gene transduced SHI-1 (SHI-1-TIMP-2) and SHI-1 transduced MSCV gene (SHI-1-MSCV) cells were inoculated via tail vein into 6-week nude mice, which pretreated by splenectomy, cytoxan intraperitoneal injection, and sublethal irradiation(referred as SCI nude mice). 30 days after inoculation, half of the mice were sacrificed, and the infiltration patterns were investigated by histological exam and human CD45 immunohistochemistry, other mice were observed for survival time. 2) Leukemic cells inoculated subcutaneously into the axillary area of mice without any pre-treatment. On day 23 and 30, mice were sacrificed to measure the volume of neoplasm. TIMP-2 protein expression and the micro vein density were detected by immunohistochemistry.

RESULTSIn SCI nude mice inoculated via caudal vein with SHI-1-TIMP-2 cells, the survival time was shorter and infiltration (including in central nervous system) was higher than that in those inoculated with SHI-1-MSCV cells. However, in inoculated subcutaneously group, the neoplasm though grew rapidly at first, over expression of TIMP-2 limited the tumor growth and angiogenesis.

CONCLUSIONThe functions of TIMP-2 are diversity; the role of TIMP-2 in tumor infiltration and metastasis was worthy of further investigation.

Animals ; Cell Line, Tumor ; DNA, Complementary ; genetics ; Genetic Vectors ; Humans ; Leukemia, Experimental ; genetics ; pathology ; Leukemic Infiltration ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Tissue Inhibitor of Metalloproteinase-2 ; genetics ; Transfection