Child ; Cytochrome P-450 CYP3A ; genetics ; Disease Susceptibility ; enzymology ; Female ; Genome-Wide Association Study ; Humans ; Male ; Polymorphism, Genetic ; genetics ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; genetics

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

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.

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

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

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 ; pharmacology ; CD11b Antigen ; genetics ; Cell Differentiation ; drug effects ; genetics ; Cell Line, Tumor ; Gene Expression Regulation, Neoplastic ; drug effects ; Humans ; Leukemia, Promyelocytic, Acute ; genetics ; pathology ; Molecular Sequence Data ; Reverse Transcriptase Polymerase Chain Reaction ; methods ; Tretinoin ; pharmacology ; Vascular Endothelial Growth Factor A ; genetics

Background and Objective:Studies on survivin over the past 5 years have shown that survivin participates in the genesis of several human cancers,including bladder cancer.Recent studies have indicated that survivin splice variants appeared to have unique subcellular localizations and functions as well.This study was to explore the roles of survivin and its two splice variants survivin-2B and survivin-△Ex3 in transitional cell carcinoma of bladder (BTCC).Methods:The relative amount of survivin,survivin-2B, and survivin-△Ex3 mRNA of fresh carcinoma tissues from 60 patients with BTCC and 12 non-cancerous bladder tissues were detected by real-time quantitative reverse transcription polymerase chain reaction(RT-PCR),and the relationships of their expression levels in different pathologic grades to clinical stages of bladder cancer were analyzed.The time of follow-up was 4-24 months.Results:Survivin,survivin-2B,and survivin-△Ex3 mRNA were detected in all BTCC tissues,and their relative expressions were 0.333±0.163,0.056±0.017, and 0.124±0.096,respectively. In the control group,three and four samples expressed survivin and survivin-△Ex3 mRNA respectively,and all samples expressed survivin-2B mRNA.The expressions of survivin and survivin-△Ex3 mRNA were positively correlated with the pathologic grades and clinical stages(0＜r'_s＜1,P＜0.05),however,survivin-2B mRNA was negativly correlated with those (-1＜r'_s＜0,P＜0.05).Conclusion:Detecting the expression levels of survivin and its two splice variants survivin-2B and survivin-△Ex3 mRNA in BTCC by real-time PCR could have potential values to evaluate tumor progression and recurrence rate.

The aim of this study was to construct venus vector carrying the gene encoding insulin-like growth factor binding protein 7 (IGFBP7), which provides an effective platform for exploring the function of this gene in leukemia. After digestion by restriction endonuclease, the IGFBP7 gene was recombined with the transfer plasmid. The venus particles were packaged using 293T cells to transfect K562 cells, and identification was performed by means of flow cytometry, RT-PCR and Western blot. The results showed that the sequence of cloned IGFBP7 gene was the same as that in GenBank. The size of product restricted by BamHI was same as the predicted one. GFP expression was observed in 293T and K562 cells with the fluorescent microscopy and flow cytometry. The expression level of mRNA and protein of IGFBP7 was confirmed by RT-PCR and Western blotting in K562 cells. It is concluded that venus vector carrying IGFBP7 gene has been successfully constructed and provides basis for exploring function of IGFBP7 in K562 cells.
Cloning, Molecular ; Gene Expression ; Genetic Vectors ; Humans ; Insulin-Like Growth Factor Binding Proteins ; genetics ; K562 Cells ; Lentivirus ; genetics ; Plasmids ; Transfection

In order to study the potential of Venus, lentiviral vector, applied to acute myeloid leukemia, the recombinant vector Venus-C3aR was transfected into 293T packing cells by DNA-calcium phosphate coprecipitation. All virus stocks were collected and transfected into HL-60, the GFP expression in HL-60 cells was measured by flow cytometry. The expression level of C3aR1 in transfected HL-60 cells was identified by RT-PCR and flow cytometry. The lentiviral toxicity on HL-60 was measured by using CCK-8 method and the ability of cell differentiation was observed. The results indicated that the transfection efficacy of lentiviral vector on HL-60 cells was more than 95%, which meets the needs for further study. C3aR1 expression on HL-60 cells increased after being transfected with recombinant lentiviral vector. Before and after transfection, the proliferation and differentiation of cells were not changed much. It is concluded that the lentiviral vector showed a high efficacy to transfect AML cells and can be integrated in genome of HL-60 cells to realize the stable expression of interest gene. Meanwhile, lentiviral vector can not affect HL-60 cell ability to proliferate and differentiate.
Genetic Vectors ; HL-60 Cells ; Humans ; Lentivirus ; genetics ; Transfection

OBJECTIVETo study the expression ratio of AML1-ETO9a (AE9a) isoform in t(8;21) acute myeloid leukemia (AML) and its clinical significance.

METHODSBone marrow samples from 44 newly diagnosed t(8;21) AML patients co-expressed AE9a and AE were screened by RT-PCR. The alteration of the AE9a expression ratio was monitored during follow-up by using quantitative real-time RT-PCR (qPCR).

RESULTSThe expression level of AE9a was markedly lower than that of AE in these patients. There was a positive correlation between the expression level of AE9a and AE in most of bone marrow samples. The transcript level of both AE9a and AE was decreased in the 44 patients after one course of standard chemotherapy, but the percentage of AE9a expression level was increased in comparison with that before treatment (P < 0.05). After one course of standard chemotherapy treatment, the percentage of AE9a in incomplete remission (ICR) patients was significantly higher than that in CR patients (P < 0.05). Relapsed patients had a higher AE9a ratio than the unrelapsed patients (P < 0.05). During the remission, the percentage of AE9a in 11/17 relapsed patients obviously elevated even while the expression of AE fusion gene at low level.

CONCLUSIONSAE9a and AE co-expressed in most of AML patients with t(8;21) translocation. The expression level of AE9a was lower than that of AE, and there is a positive correlation between the expression level of these two isoforms. The sensitivity of AE9a gene to the standard chemotherapy is less than that of the AE fusion gene. Monitoring the AE9a to AE ratio during the CR can predict the early relapse of the disease compared to monitoring the AE alone.

Adolescent ; Adult ; Child ; Chromosomes, Human, Pair 21 ; Chromosomes, Human, Pair 8 ; Core Binding Factor Alpha 2 Subunit ; genetics ; Female ; Gene Expression ; Humans ; Leukemia, Myeloid, Acute ; genetics ; pathology ; Male ; Middle Aged ; Oncogene Proteins, Fusion ; genetics ; Protein Isoforms ; genetics ; RUNX1 Translocation Partner 1 Protein ; Translocation, Genetic ; Young Adult