Clinical analysis of 12 cases of acute myeloid leukemia with Ph chromosome and BCR-ABL positive / 中国实验血液学杂志

This study was purposed to analyze the characteristics of morphology, immunology, cytogenetic and molecular biology of leukemia cells in 12 AML patients with Ph(+) and their correlation with survival of patients. 12 patients with Ph(+) AML were diagnosed according to diagnostic criteria of WHO and existence of t(9;22) (q34;q11) or t(9;22) abnormality, meanwhile no evidence of CML chronic phase was observed. The results showed that 8 out of 12 cases were confirmedly diagnosed to be AML by morphologic and immunophenotypic examination, 4 cases were diagnosed as myeloid and B lymphocytic mixed acute leukemia. The Ph chromosome was detected in 10 cases by chromosome analysis at the first time of diagnosis, and some of the cases had coexistence of complex chromosome and/or normal karyotype. BCR-ABL transcript was detected in all 12 cases, including 7 cases with b3a2, 1 case with b2a2, 1 case with b2a2 variants, 2 cases with e1a2 and 1 case with e18a2. The 12 cases all got complete remission after chemotherapy and/or gleevec treatment, out of them 3 cases received chemotherapy and gleevec treatment, but 2 cases died; 9 cases received allogeneic hematopoietic stem-cell transplantation (allo-HSCT), 1 case died from relapse, among them 1 case died from transplant complications. The median survival was 24 (8 - 80) months, the overall survival of 3 years was (51.4 ± 17.7)%. It is concluded that the Ph(+) AML is a acute myelogenous leukemia with poor prognosis, but long-term survival may be achieved with HSCT as quick as after complete remission from gleevec and chemotherapy treatment. Meanwhile, the detection of BCR-ABL gene and it variants may be give more opportunity for diagnose and treatment, which can be used as routine screening for newly diagnosed leukemia.

Detecting PML-RARalpha transcript in acute promyelocytic leukemia using real-time quantitative RT-PCR / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Real-time quantitative RT-PCR (RQ-PCR) assay has become a vital tool to monitor residual disease of leukemia. However, the complexity and standardization of RQ-PCR should never be overlooked and the results should be interpreted cautiously in clinical conditions. We aimed to assess the methodology of RQ-PCR and its clinical applications in monitoring molecular kinetics of 36 newly diagnosed cases of acute promyelocytic leukemia patients with t (15; 17) from October 2004 to December 2005.</p><p><b>METHODS</b>All the TaqMan probe-based RQ-PCR reactions and analysis were performed on an ABI-PRISM 7,500 platform. The quantitation of PML-RARalpha transcripts was represented by the normalized quotient, that is, PML-RARalpha transcript copies divided by ABL transcript copies. According to induction therapy, the patients were classed into two groups: group 1 (n = 23), three-drug combination including arsenics, all-trans retinoic acid and mitoxantrone; and group 2 (n = 13), two-drug combination from all-trans retinoic acid, arsenics and mitoxantrone.</p><p><b>RESULTS</b>The sensitivity of RQ-PCR was 1 per 10(5) cells and 5 copies of the PML-RARalpha transcript could be reproducibly detected. No false positive results occurred in 40 non-acute promyelocytic leukemia samples. Optimal amplification efficiency could be attained, which was determined by the slope of the standard curves (slope: -3.2 - -3.7). The inter-assay and intra-assay variation coefficients of the method were 1.01% and 0.56% respectively. Although the time to attain hematological complete remission was similar in both groups, the time to achieve molecular remission of group 1 was significantly shorter than that of group 2 (61 days vs 75 days, P = 0.034). The rate of molecular remission within 70 days was higher in group 1 than in group 2 (75.00% vs 38.46%, P = 0.036). Compared with pretreatment, median reduction of the PML-RARalpha transcript before first consolidation therapy differed significantly between group 1 and group 2 (log scale, 3.15 vs 2.31, P = 0.024). Interestingly, we found that PML-RARalpha transcript levels temporarily increased in bone marrow (7 patients) and peripheral blood (22 patients) samples of patients during induction therapy in both groups.</p><p><b>CONCLUSIONS</b>The RQ-PCR assay is reliable for the detection of PML-RARalpha transcripts. Arsenics, all-trans retinoic acid and mitoxantrone triad induction treatment of acute promyelocytic leukemia is superior to two-drug combination induction therapy in terms of the molecular response.</p>

Effect of the new human transcription factor hBKLF on the proliferation, differentiation of K562 cell line and hemoglobin synthesis / 中国实验血液学杂志

The human basic Krüppel-like factor (hBKLF) is a newly cloned human transcription factor from the cDNA library of fetal liver. It belongs to the Krüppel-like transcription factor family. Previous expression study showed that it is a hematopoietic related factor. This study was aimed to investigate the effect of hBKLF on cell proliferation, differentiation and hemoglobin synthesis by using K562 cell line as model. The sense and antisense expression plasmids of hBKLF were constructed, and transfected into K562 cells by lipofectamine. After G418 selection for 4 weeks, the cell line with stable expression of the gene was obtained. Then the hBKLF expression level, proliferation ability, colony formation and hemoglobin production were detected by RT-PCR and Western blot, MTT method, methyl cellulose semisolid culture method and benzidine test respectively. The morphologic change of cell was observed with inverted microscope. The results showed that the sense plasmid could increase hBKLF level and antisense plasmid could decrease hBKLF expression. When hBKLF level was down-regulated, K562 cells could proliferate more quickly and synthesize more hemoglobin. But there were no differences in colony formation ability and no apparent morphologic change. It is concluded that hBKLF can inhibit hematopoietic cell proliferation and hemoglobin synthesis. It is suggested that hBKLF plays an important role in the proliferation and differentiation of hematopoietic cells.

Methylation status of multidrug resistance (mdr1) gene and its correlation with expression of mdr1 gene in patients with hematologic malignancies / 中国实验血液学杂志

To investigate the correlation between methylation and expression of multidrug resistance (mdr1) gene, restriction endonuclease HpaII combined with competitive PCR technique was used to quantitatively detect the methylation status of two CCGG sites located at -110 and -50 bp (region I and II) up to the transcription start site in mdr1 promoter in 54 AL and 9 MM patients. Semi-quantitative RT-PCR was used to detect the expression level of mdr1 gene. The results showed that inverse correlation between methylation rate of either region or total methylation rate and expression of mdr1 gene was observed. The correlation in the region I (r = -0.64) was closer than that in the region II (r = -0.4). High expression rate of mdr1 ascended significantly in low methylation group (n = 36) (P < 0.001). In comparison with chemotherapy sensitive group (n = 8), the methylation rate in refractory AL patients (n = 16) was lower (P = 0.05) in the region I, P < 0.05 in the region II and total regions. Comparing with the untreated patients (n = 36), the methylation rate in the region I and total methylation rate were lower in the patients with chemotherapy (n = 14) (P < 0.05). The methylation rate in the region II was also decreased after chemotherapy, however, no statistical significance was shown (P > 0.05). Increased mdr1 expression level accompanying with decreased methylation rate after chemotherapy was found, although no significant difference was shown (P = 0.06). It is concluded that the expression level of mdr1 gene was associated with the methylation status of CCGG in -110 and -50 bp upstream to the transcription start site, especially the -110 site. In both the patients treated with chemotherapy and the refractory patients, the methylation level of mdr1 gene decreased relatively. The rising expression of mdr1 gene after chemotherapy was associated with the decrease of methylation level.

Reversal effect of berbamine on multidrug resistance of K562/A02 cells and its mechanism / 中国实验血液学杂志

This preject is to explore the reversal efficacy of calmodulin antagonist berbamine (BBM) on multidrug resistance (MDR) and its mechanism. Human erythroleukemic cell line K562 and its adriamycin-resistant counterpart K562/A02 were used in the study. The cells were co-cultured with ADR and BBM in different concentrations. MTT assay was used to analyze the effect of BBM on cell growth inhibition. According to the MTT assay, the 50% inhibitory concentration (IC(50)), the multiples of drug resistance and increased sensitivity of ADR were calculated. The concentration of intracellular ADR and expression level of P-gp were detected by flow cytometry (FCM). The expression level of mdr1 mRNA and survivin mRNA was detected by semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) with beta-actin as internal reference. The results showed that IC(50) of ADR in K562 and K562/A02 cells was 1.16 +/- 0.09 micro mol/L and 37.47 +/- 1.76 micro mol/L, respectively. The resistant multiple of K562/A02 cells to ADR was 32.30 higher than that of K562 cells. BBM increased the chemo-sensitivity of ADR in K562/A02 cells with dose-dependent relationship, i.e. when 5, 10 and 20 micro mol/L BBM was added in the culture the chemo-sensitivity of ADR was increased to 2.01-, 9.68-, and 41.18-fold (P < 0.01), respectively. After treating K562/A02 cells by 5 or 10 micro mol/L BBM for 2 hours the accumulation of intracellular ADR was increased to 1.41- and 1.52-fold (P < 0.01), respectively. Treating by BBM for 72 hours decreased 4.12% (P < 0.05) and 27.09% (P < 0.01) of P-gp expression, respectively, meanwhile down-regulated expression of mdr1 mRNA and survivin mRNA was found. In conclusion, BBM could increase intracellular concentration of ADR in K562/A02 that down-regulated expression level of mdr1 mRNA and P-gp and survivin so that the sensitivity of K562/A02 to ADR was increased significantly.

Inhibitory effect on proliferation of KG1a cell line by methyltransferase inhibitors / 中国实验血液学杂志

To explore the possibility of a new therapeutic strategy for leukemia by intervening in the DNA methylation to re-express p15 suppressor gene, methylation inhibitors, 5-Aza-2'-deoxycytidine (5-Aza-CdR) and cell differentiation agent (CDAII) were used to treat myelogenous leukemia cell line KG1a in which p15 gene expression was suppressed due to DNA hypermethylation. The biological characteristics of KG1a cells untreated or treated with the agents were investigated and analyzed using morphology, methylation specific-PCR (MSP), (3)H-labeled microassay technique, restriction endonuclease reaction, flow cytometry and immunofluorescence methods. The results indicated that both agents showed concentration-dependent and time-dependent inhibition of cell proliferation. 5-Aza-CdR and CDAII induced apoptosis and cell differentiation with G(2) and G(0)/G(1) arrest respectively. Furthermore, DNA methyltransferase activity and level of methylation in genomic DNA were decreased and p15 protein was re-expressed partially. It is concluded that it is possible to treat leukemia by intervening in the DNA methylation using methyltransferase inhibitors and it is worth to make a thorough study on mechanism of the new strategy.