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

To elucidate the possible mechanism of differentiation and/or apoptosis in NB4, K562 cells induced by tributyrin (TB), a histone deacetylase inhibitor (HDACi), the level of acetylated histone H3 was detected by Western blot, p21(WAF1) expression was detected by semi-quantitative RT-PCR. The results showed that histone H3 hyperacetylation was detected in NB4 (or K562) cells after treatment with TB 0.1 mmol/L (or TB 0.5 mmol/L) for 16 hours in a dose-dependent manner. p21(WAF1) dose-dependently increased at RNA level in these two cell lines treated by TB 0.1 mmol/L. The level of p21(WAF1) mRNA increased at 2 hours after TB treatment, reaching peak at 16 hours, and maintaining for 48 hours. In conclusion, the mechanism of differentiation and apoptosis in NB4, K562 cells induced by tributyrin may associate with its up-regulation of acetylated histone and p21(WAF1) mRNA level.
Acetylation ; drug effects ; Apoptosis ; drug effects ; Blotting, Western ; Cell Differentiation ; drug effects ; Cell Line, Tumor ; Cyclin-Dependent Kinase Inhibitor p21 ; genetics ; Dose-Response Relationship, Drug ; Enzyme Inhibitors ; pharmacology ; Gene Expression Regulation, Neoplastic ; drug effects ; Histone Deacetylase Inhibitors ; Histone Deacetylases ; metabolism ; Histones ; metabolism ; Humans ; K562 Cells ; Leukemia ; genetics ; metabolism ; pathology ; RNA, Messenger ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Triglycerides ; pharmacology

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 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