Promoter methylation status of PTEN gene and the effect of induced demethylation in leukemia cell lines / 中华血液学杂志

<p><b>OBJECTIVE</b>To explore the mechanism of PTEN gene expression silence in leukemia cells, and the effect of induced PTEN gene expression in leukemia cells.</p><p><b>METHODS</b>Methylation status of PTEN in leukemic cell lines, including HL-60, Nalm-6, NB4, U937, Raji, K562 and KG-1a was assessed by methylation specific PCR (MSP). The cell lines were then treated with different concentrations of methyltransferase inhibitor 5-Aza-2'-deoxycytidine (5-Aza-CdR). After that the changes in PTEN methylation status were detected by MSP, and PTEN mRNA expression level by reverse transcription PCR (RT-PCR). Growth inhibition and apoptosis of HL-60 and Nalm-6 cells induced by 5-Aza-CdR were observed by MTT assay, and Wright and Annexin V staining, respectively.</p><p><b>RESULTS</b>Hypermethylation of PTEN promoter was detected in HL-60, U937, Nalm-6, Raji and KG-1a, while hypomethylation was found in NB4 and K562 by MSP. After 5-Aza-CdR treatment, the hypermethylation status of PTEN promoter in HL-60 and Nalm-6 cells was reversed and their PTEN mRNA expression levels were up regulated in dose dependent manner with the 5-Aza-CdR concentrations, and the cell apoptosis was induced.</p><p><b>CONCLUSION</b>Hypermethylation in the promoter region is one of major mechanisms responsible for transcriptional suppression of PTEN. Methyltransferase inhibitor could induce the expression of PTEN gene and lead to the leukemia cells apoptosis.</p>

The expression of midkine in acute leukemia and its significance / 中华血液学杂志

<p><b>OBJECTIVE</b>To analyze the expression of midkine (MK) gene in acute leukemia patients, and explore the relationship between the gene and leukemia.</p><p><b>METHODS</b>The MK gene expression levels were detected by real-time quantitative RT-PCR (RQ-RT-PCR) in bone marrow (BM) of 181 acute leukemia (AL) patients and 31 normal controls.</p><p><b>RESULTS</b>MK gene was expressed in all AL patients, normal controls and AL patients in complete remission (CR). Compared with that in control group and CR group, MK gene expression was significantly increased in AL patients (P < 0.01 and P < 0.05, respectively). No statistical difference was found between CR group and control group. The expression of MK showed a notable increase in all B-ALL subtypes (including pro-B-ALL, common-B-ALL and pre-B-ALL) as well as in adult and childhood B-ALL patients (P < 0.01). Moreover, the gene expression in B-ALL was also significantly higher than that in TALL, HAL and FAB subtypes of AML (P < 0.01). In addition, M2 patients showed significantly increased in MK expression compared with that in normal controls (P < 0.01) and in other FAB subtypes of AML (P < 0.05). Median MK expression level in M3 patients was also significantly higher than that in normal controls (P < 0.05), but there was no statistical difference between M3 and other AL subtypes excepting for M2 and B-ALL. MK expression in CD34 positive patients was significantly higher than that in CD34 negative ones (P < 0.01) and within M2 patients, MK expression was higher in patients with t (8 ;21) than in those without the translocation (P < 0.05).</p><p><b>CONCLUSION</b>MK gene expression is increased with different levels in B-ALL, M2 and M3 patients, which provides novel insights into the leukemogenesis of acute leukemia.</p>

Values of impulse oscillometry for pulmonary function and adhesion molecules in childhood asthma / 中国当代儿科杂志

<p><b>OBJECTIVE</b>Impulse oscillometry (IOS) is a novel technique for the evaluation of pulmonary function. Soluble intercellular adhesion molecule-1 (sICAM-1) and soluble vascular cell adhesion molecule-1 (sVCAM-1) are definitive indicators for the severity of asthma. This study aimed to explore the relationship of IOS pulmonary function with sICAM-1 and sVCAM-1, and their values in childhood asthma.</p><p><b>METHODS</b>IOS via Master Screen System for pulmonary function was performed in 40 children with acute asthma and 25 healthy children. Twenty-three of 40 children with acute asthma were re-tested for IOS pulmonary function at remission. sICAM-1 and sVCAM-1 levels were measured in 23 children with acute asthma, 20 asthmatic children at remission and 16 healthy children.</p><p><b>RESULTS</b>The parameters of IOS pulmonary function, R5, R20, R5-R20, X5, Fres and Zrs in children with acute asthma were significantly higher than in asthmatic children at remission and in normal controls (q= 2.91-15.61, P < 0.01 or 0.05). There were significant differences in R5, R5-R20, Fres and Zrs between the asthmatic children at remission and normal controls (q= 3.08- 9.19, P < 0.01 or 0.05). sICAM-1 and sVCAM-1 levels in children with acute asthma were significantly higher than in asthmatic children at remission and in normal controls (q= 6.23-26.15, P < 0.01). The asthmatic children at remission had higher levels of sICAM-1 and sVCAM-1 than the normal controls (q=16.86, 12.46, P < 0.01). R5-R20 positively correlated with sICAM-1 and sVCAM-1 in children with acute asthma (r=0.45, 0.57, P <0.05).</p><p><b>CONCLUSIONS</b>IOS for pulmonary function and sICAM-1 and sVCAM-1 may be used to evaluate the severity and therapeutic effects of childhood asthma. A correlation exists between IOS pulmonary function and sICAM-1 and sVCAM-1.</p>

Effects of AML1-ETO on transcription activity of p21WAF1/CIP1 gene promoter / 中华血液学杂志

<p><b>OBJECTIVE</b>To observe the effects of AML1-ETO fusion gene on the transcription activity of p21WAF1/CIP1 gene. And to explore the enhancement of leukemia pathogenesis of AML1-ETO.</p><p><b>METHODS</b>The luciferase reporter plasmids of p21WAF1/CIP1 gene promoter were constructed, and co-transfected into CV-1 cells with AML1-ETO, AML1b and AML1a expression plasmids. The trans-activity of p21WAF1/CIP1 gene promoter was assayed by luminometer.</p><p><b>RESULTS</b>AML1-ETO exhibited a distinct inhibition activity of p21WAF1/CIP1 gene promoter with a sequence-specificity and dosage-dependent manner. The trans-activity of p21WAF1/CIP1 gene promoter decreased to (19 +/- 4)% compared to control group, when 1000 ng pCMV5-AML1-ETO plasmid was used. AML1b and AMLla showed less inhibition activity. The trans-activity of p21WAF1/CIP1 gene promoter decreased to (61 +/- 16)% and (59 +/- 16)% compared to control group, respectively, when 1000 ng plasmid was used.</p><p><b>CONCLUSION</b>AML1-ETO exhibits more inhibition activity of p21WAF1/CIP1 gene promoter than AML1b and AMLla, results from recruiting transcription co-repression complex efficiently by ETO. Based on previous researches, the effects of exogenous AML1-ETO on p21WAF1/CIP1 gene promote may be dependent on the type of cell lines.</p>

Phenol butyrate inhibits cell cycle of leukemia cell lines through up-regulation of p21WAF1/CIP1 gene / 中华血液学杂志

<p><b>OBJECTIVE</b>To elucidate effects of histone deacetylase inhibitors on cell cycle of leukemia cell lines and investigate its molecular mechanisms.</p><p><b>METHODS</b>Kasumi-1, U937 and NB4 cell lines were exposed to a histone deacetylase inhibitor, phenyl butyrate (PB), for 24, 48 and 72 hrs. Cells were harvested for cell cycle analysis by flow cytometry. Gene expression of p21WAF1/CIP1, a cyclin-dependent kinase inhibitor, was determined by semi-quantitative reverse transcriptase polymerase chain reaction (semi-quantitative RT-PCR). Promoter activity of p21WAF1/CIP1 was determined by luciferase-reporter assay in 293T cell line.</p><p><b>RESULTS</b>PB inhibited cell cycle of Kasumi-1, U937 and NB4 cell lines, showing G(0)/G(1) phase arrest and S-phase fraction reduction with a dose and time dependent manner. After Kasumi-1, U937 or NB4 cells exposed to 3 mmol/L PB for 72 hrs, G(0)/G(1)-phase fraction was increased by 42.03%, 44.36% and 26.82%, and S-phase fraction was decreased by 31.86%, 38.9% and 26.77%, respectively. After Kasumi-1, U937 and NB4 cell lines exposed to PB, the expression of p21WAF1/CIP1 gene was increased by (2.06 +/- 0.27), (2.78 +/- 0.40) and (1.78 +/- 0.20) times at its maximum, respectively. PB could stimulate p21WAF1/CIP1 promoter activity (by luciferase-reporter assay) and the effect was dose dependent. The promoter activity was increased by 5.74 times after the cells exposed to 3 mmol/L PB for 48 hrs. PB stimulating p21WAF1/CIP1 promoter activity was mainly mediated by a 101 base pairs fragment upstream of transcription start site.</p><p><b>CONCLUSION</b>PB could inhibit cell cycle of leukemia cell lines. The effects were mainly through up-regulation of p21WAF1/CIP1 expression.</p>

The heat shock protein 90 inhibitor induces apoptosis and differentiation of Kasumi-1 and its mechanisms / 中华血液学杂志

<p><b>OBJECTIVE</b>To explore the effect of 17-allylamide-17-demethoxygeldanamycin (17AAG), a heat shock protein 90 (HSP90) inhibitor, on the growth, differentiation and apoptosis of leukemic Kasumi-1 cells.</p><p><b>METHODS</b>Kasumi-1 cells were treated with 17AAG at different concentrations in suspension culture. Cell proliferation was analysed by MTT assay, expression of myeloid-specific differentiation antigen and cell cycle by flow cytometry, cell apoptosis by annexin V staining, agarose gel electrophoresis and flow cytometry. KIT protein was analysed by Western blot and c-kit mRNA by RT-PCR.</p><p><b>RESULTS</b>17AAG treatment caused a dose-dependent inhibition of the cell proliferation with the IC(50) of 0.62 micromol/L. A dose-dependent increase in early apoptosis occurred at 24 hours treatment and in late apoptosis at 48 hours treatment. 17AAG induced a time- and dose-dependent increase in expression of myeloid cell surface protein CD11b and CD15, a progressive decline in S-phase cell fraction and an increase in G(0)/G(1) cells. When Kasumi-1 cells were incubated with 1 micromol/L of 17AAG, KIT protein began to decrease at 2 hours and KIT protein could hardly be detected at 20 hours, but c-kit mRNA was not decreased.</p><p><b>CONCLUSION</b>17AAG treatment of Kasumi-1 cells could lower KIT protein expression, inhibit cell proliferation, induce cell partial differentiation, apoptosis and accumulation in G(0)/G(1) phase.</p>