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

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

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

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.

To elucidate the effect of established primary bone marrow stromal layers on the gene transduction of human hematopoietic stem/progenitor cells (HSC/HPC), mononuclear cells (MNC) from adult bone marrow were isolated by centrifugation on Ficoll-Hypaque gradients and plated in stromal culture medium. The cells were incubated until passage 4 to establish primary stromal layers. The HSC/HPC prestimulated by cytokines were transduced by retroviral supernatant containing mdr1 gene in presence of irradiated stroma-contact support. Transduced cells were plated in a colony-forming unit assay with and without vincristine (VCR) to assess the efficiency of transduction. Individual colonies were also analyzed by polymerase chain reaction (PCR) for the presence of provirus. The results showed that the mixed adherent cell layers were formed when adult bone marrow stromal cells were incubated for four to six weeks, mainly being composed of fibroblasts. In the presence of stroma-contact support, the average of gene transduction efficiency in marrow-derived progenitors increased 2.1 to 3.3 folds measured by colony-forming assay and/or PCR, significantly higher than those without support of stroma. It is concluded that the presence of bone marrow stroma support in combination with cytokine facilitates augmenting the extent of retroviral-mediated gene transduction.
Bone Marrow Cells ; physiology ; Genes, MDR ; Hematopoietic Stem Cells ; metabolism ; Humans ; Retroviridae ; genetics ; Stromal Cells ; physiology ; Transduction, Genetic

OBJECTIVETo explore the effect of down-regulation of insulin-like growth factor binding protein 7 (IGFBP7) on the proliferation and invasiveness of leukemia cell line U937 cells.

METHODSThree pairs of double-strand siRNA targeting IGFBP7 gene were transfected into SMMC7721 cells to select the most efficient one for U937 cells. qRT-PCR and Western blot were used to detect the expression of IGFBP7 in U937 cells after transiently transfected with siRNA of IGFBP7. Cell proliferation, adhesion, trans-endothelial migration and invasion were performed in transfected cells and control groups.

RESULTSAfter transfected with siRNA of IGFBP7 in U937 cells, the ability of cell proliferation was significantly decreased at 24 h (0.580 ± 0.159) compared to that of parental cells and scramble negative control (1.049 ± 0.274, 0.946 ± 0.195, respectively) (P < 0.01). Adhesion of U937 cells transfected with IGFBP7 gene specific siRNA to ECV304 cells was significantly lower than that of the control groups (0.247 ± 0.031 vs 0.406 ± 0.023 and 0.395 ± 0.011) (P < 0.01). Transendothelial membrane of U937 cells into the bottom of the 24-well plate for experimental group were less than those in the control groups \[(0.387 ± 0.021)×10(5) vs (1.017 ± 0.031)×10(5) and (0.908 ± 0.027)×10(5)\]. Cells adherent to the matrigel for experimental group were less than those in the control groups \[(0.197 ± 0.098)×10(5) vs (0.493 ± 0.067)×10(5) and (0.469 ± 0.083)×10(5)\]. The difference was significant (P < 0.01).

CONCLUSIONIGFBP7 gene plays a contributing role in leukemogenesis involving in leukemic cells' proliferation and interaction with endothelial cells through adhesion, invasion and migration.

Cell Proliferation ; Down-Regulation ; Humans ; Insulin-Like Growth Factor Binding Proteins ; genetics ; metabolism ; Leukemia, Myeloid, Acute ; metabolism ; pathology ; Transfection ; U937 Cells

cDNA microarray, recently applied to analyze gene expression profile of cancers, was difficult to be utilized in myelodysplastic syndrome (MDS) for a special need of excessive mRNA hardly provided by ordinary bone marrow aspiration in MDS patients. The aim of this study was to investigate the feasibility of exploring the molecular events underlying MDS by using cDNA microarray and mixing mRNA from multiple patients. A commercially purchased BioStar H141 cDNA microarray containing 14,110 clones of cDNA or EST was employed to analyze the gene expression profile of bone marrow mononuclear cells from two cases of MDS. Equal amount of total RNA from each patient was mixed, reversely transcribed to cDNA and labeled with Cy5. Mixture of Cy5-labeled cDNA and Cy3-labeled cDNA from normal bone marrow cells was concomitantly hybridized to H141 microarray in duplicate. In H141 chips, 1,064 cDNAs were spotted at least twice targeting different fragments of a single gene cDNA. The results showed that among these 1,064 cDNA clones, the expression level of 625 (58.7%) and 630 (59.2%) ones was consistent within these two chips, respectively, 297 (27.9%) and 191 (18.0%) inconsistent, 21 (2.0%) and 11 (1.0%) in opposite. Among 411 duplicately spotted cDNAs with complete data, expression levels of 400 (97.3%) was consistent between two chips. 488 genes with known function were identified as differentially expressed in MDS, among which 101 genes were involved in hematopoiesis regulation, including those encoding transcription factors, cell cycle-regulating proteins, metabolism-relating enzymes, and adhesive molecules. It is concluded that cDNA microarray can be used for profiling gene expression of mixed MDS samples and replication shall be necessary for reducing the data bias caused by experimental operation.
Bone Marrow Cells ; metabolism ; Gene Expression Profiling ; Humans ; Leukocytes, Mononuclear ; metabolism ; Male ; Middle Aged ; Myelodysplastic Syndromes ; metabolism ; Oligonucleotide Array Sequence Analysis ; Reproducibility of Results