2.Expression of WT1 Gene in Acute Leukemia in Children and Its Clinical Significance
xu-li, WANG ; yi-huan, CHAI ; shao-yan, HU ; jian-nong, CEN
Journal of Applied Clinical Pediatrics 1993;0(03):-
Objective To explore the expression of WT1 gene in acute leukemia in children and its clinical significance.Methods The real-time quantitative reverse transcription-polymerase chain reaction method was used to detect the expression level of WT1 gene in 198 children with acute leukemia.Results The medium of WT1 gene in children with acute leukemia was 932.99,but it was 38.50 in control group,and it in patient′s group was significantly higher than that in control group.The medium of WT1 gene in children with ALL was 195.73,while the medium of WT1 gene in children with acute myeloid leukemia was 6 297.75,and there was significant difference between the 2 groups(P
3.Examination of point mutations within the ABL kinase domine region of BCR/ABL in patients with chronic myeloid leukemia who develop imatinib resistance
Dong-Guang YANG ; Ri ZHANG ; Jian-Nong CEN ; Zi-Ling ZHU ;
Chinese Journal of Laboratory Medicine 2003;0(11):-
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.
4.Relationship between Runx3 gene expression and its DNA methylation in gastric cancer.
Nan GAO ; Wei-Chang CHEN ; Jian-Nong CEN
Chinese Journal of Oncology 2008;30(5):361-364
OBJECTIVEThis study was designed to clarify the significance of DNA methylation in the expression of runt-related transcription factor 3 (Runx3) gene.
METHODSReverse transcription-PCR (RT-PCR) was used to measure the expression level of Runx3 mRNA in paired samples of primary gastric cancer and corresponding non-cancerous gastric mucosa, taken from surgical specimens of 70 gastric cancer patients. Western blot was used to detect the protein expression level of Runx3 gene. The promoter methylation status of Runx3 gene was detected by methylation specific PCR (MSP). Furthermore, RT-PCR was used to mesure the expression of DNA methyltransferase 1 (Dnmtl) mRNA . The correlation of Runx3 expression and methylation with Dnmt1 mRNA expression was analyzed.
RESULTSThe mRNA expression level of Runx3 gene was significantly lower in gastric cancer than that in the matched normal gastric mucosa (0.5740 +/- 0.3580 vs. 1.7250 +/- 0.4080, P < 0.05), and the Runx3 protein expression level in gastric cancer was also significantly lower than that in the matched normal gastric mucosa (P < 0.05). Promoter hypermethylation of Runx3 gene was detected in 50.0% (28/56) of the gastric cancer samples, which resulted in a reduced expression of Runx3 mRNA. It was found that the mRNA expression level of Dnmt1 gene was significantly higher in the gastric cancer tissues with methylated Runx3 gene than that in the ones without. There was a significant correlation of Runx3 gene methylation with increased expression of Dnmtl mRNA (r = 0.64, P < 0.05).
CONCLUSIONThe promoter hypermethylation may be one of the predominant inactivation mechanisms of the runt-related transcription factor 3 gene, and may be associated with carcinogenesis of human gastric cancer. Reduced Runx3 expression in gastric cancer may be partially correlated with a high level of DNA methyltransferase 1.
Adenocarcinoma ; genetics ; metabolism ; Adult ; Aged ; Core Binding Factor Alpha 3 Subunit ; genetics ; metabolism ; DNA (Cytosine-5-)-Methyltransferase 1 ; DNA (Cytosine-5-)-Methyltransferases ; biosynthesis ; genetics ; DNA Methylation ; Down-Regulation ; Female ; Gene Expression Regulation, Neoplastic ; Humans ; Male ; Middle Aged ; Promoter Regions, Genetic ; RNA, Messenger ; metabolism ; Stomach Neoplasms ; genetics ; metabolism ; Young Adult
5.Effects of tributyrin on SHI-1 leukemia cells in vitro.
Hong YIN ; Zi-Xing CHEN ; Jian-nong CEN ; Wei-ming DUAN ; Wei WANG ; Jian-xin FU
Chinese Journal of Hematology 2004;25(11):662-665
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
6.Quantitative analysis of gene expression for vascular endothelial growth factor and its application.
Xia BAI ; Jian-Xin FU ; Kai-Yang DING ; Jian-Nong CEN ; Wei WANG ; Chang-Geng RUAN
Journal of Experimental Hematology 2005;13(4):548-552
Vascular endothelial growth factor (VEGF), a central mediator of angiogenesis, not only plays an important role in the pathogenesis of leukemia, but also is an independent prognostic factor in patients with hematologic malignancies, like those in solid tumors. However, the importance of VEGF during differentiation or apoptosis of leukemia cells remains to be elucidated. In order to assess the alternation of VEGF gene expression in the process of all-trans retinoic acid (ATRA)-induced differentiation of NB4 acute promyelocytic leukemia cell line, and a competitor DNA fragment, VEGF gene competative template (T-VEGFDelta) was constructed by using gene recombinant technologies, and a competitive quantitative reverse transcriptase-polymerase chain reaction (cQRT-PCR) method was developed. A standard curve was obtained by co-amplification of serial dilutions of the target nulecules with constant amount of competitive template and this curve was used to detect molecular number of target gene in measuring sample. The surface expression of CD11b antigen and nitroblue tetrazolium (NBT) reduction rate of NB4 cells were also assayed at different time points. The results showed that cQRT-PCR was a sensitive, reliable tool for analysis of VEGF gene expression with a detectable range from 1 x 10(4) to 2 x 10(5) molecules. The number of VEGF gene transcripts detected by means of cQRT-PCR assay was 42.3 x 10(5), 12.6 x 10(5), 3.6 x 10(5), and less than 1.0 x 10(5)/microg total RNA at 0, 12, 24 and 48 hours after ATRA treatment, respectively. This rapid down-regulation of VEGF gene expression, during ATRA-induced NB4 cell differentiation, was accompanied by the up-regulation of CD11b expression and an increased NBT reduction rate. In conclusion, cQRT-PCR method was successtully constructed, confirming that ATRA efficiently repressed VEGF, at the same time, the ATRA might exert an antileukemic effect, other than induction of differentiation via inhibition of angiogenesis.
Antineoplastic Agents
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pharmacology
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CD11b Antigen
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genetics
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Cell Differentiation
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drug effects
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genetics
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Cell Line, Tumor
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Gene Expression Regulation, Neoplastic
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drug effects
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Humans
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Leukemia, Promyelocytic, Acute
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genetics
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pathology
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Molecular Sequence Data
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Reverse Transcriptase Polymerase Chain Reaction
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methods
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Tretinoin
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pharmacology
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Vascular Endothelial Growth Factor A
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genetics
7.Disruption of blood brain-barrier by leukemic cells in central nervous system leukemia.
Sa-ran FENG ; Zi-xing CHEN ; Jian-nong CEN ; Hong-jie SHEN ; Yuan-yuan WANG ; Li YAO
Chinese Journal of Hematology 2011;32(5):289-293
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
8.Growth and infiltration of human monocytic leukemia cell in nude mice: a model for central nervous system leukemia.
Zhen-jiang LI ; Zi-xing CHEN ; Jun LU ; Jian-nong CEN ; Jun HE ; Ling-chuan GUO
Chinese Journal of Hematology 2006;27(6):374-378
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
9.Preliminary establishment of transplanted human chronic myeloid leukemia model in nude mice.
Xian-Min LI ; Xin DING ; Long-Zhen ZHANG ; Jian-Nong CEN ; Zi-Xing CHEN
Journal of Experimental Hematology 2011;19(6):1378-1382
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
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Disease Models, Animal
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Humans
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Leukemia, Myelogenous, Chronic, BCR-ABL Positive
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Male
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Mice
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Mice, Nude
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Mice, SCID
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Neoplasm Transplantation
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Neoplastic Stem Cells
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Transplantation, Heterologous
10.Quantitative study on C/EBP zeta gene transcripts in patients with chronic myeloid leukemia using real-time quantitative PCR.
Jun QIAN ; Zi-xing CHEN ; Jiang LIN ; Shao-yan HU ; Jian-nong CEN ; Wei WANG
Chinese Journal of Medical Genetics 2005;22(6):628-631
OBJECTIVETo evaluate the significance of quantitative detection of CCAAT/enhancer binding protein zeta (C/EBP zeta) transcripts in patients with chronic myeloid leukemia (CML).
METHODSReal-time quantitative polymerase chain reaction (RQ-PCR) assay was established and performed to measure the transcript level of C/EBP zeta in the bone marrow mononuclear cells from 76 patients with CML at different stages.
RESULTSC/EBP zeta transcripts were significantly decreased in CML patients in chronic phage, accelerated phage, and blastic crisis (median 2.5, 3.31, and 2.22, respectively), compared to that in normal controls (median 12.20). Further down-regulation of C/EBP zeta was observed in the patients resistant to interferon (median 1.56); however, its expression level returned to normal in the patients who obtained complete cytogenetic remission (median 15.43).
CONCLUSIONC/EBP zeta was down-regulated in CML patients, which might play a role in the leukemogenesis.
CCAAT-Enhancer-Binding Proteins ; genetics ; Humans ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; methods ; Transcription, Genetic