[Clinical analysis of 12 cases of acute myeloid leukemia with Ph chromosome and BCR-ABL positive].

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.

[FIP1L1/PDGFRalpha fusion gene-negative chronic eosinophilic leukemia with t(5;12)(q31;p13): a case report and review of literatures].

OBJECTIVE: To deepen the understanding of chronic eosinophilic leukemia (CEL). METHODS: The course of diagnosis and treatment in a case of FIP1L1/PDGFRalpha fusion gene negative CEL was reported. Flow cytometry was used to analyze the immunophenotype of the cells in peripheral blood and pleural fluid. Karyotype was analyzed with G-banding. The expression of FIP1L1/PDGFRalpha fusion gene was detected by RT-PCR technique. Routine pathological examination of the tissues from bone marrow, lung and spleen were performed. RESULT: A sixteen-year-old girl had severe anemia, fever, splenomegaly, thrombocytopenia and dominant hypereosinophilia lasting for 22 months. Trephine biopsy showed a hypercellular marrow with eosinophilic proliferation and moderate reticular fibrosis. Eosinophilic infiltration was found in lung and spleen and embolism was also found in spleen. She had a clonal chromosomal abnormality t(5;12)(q31;p13). The expression of FIP1L1/PDGFRalpha was negative. An abnormal clone of T cells expressing CD(3)(-), CD(4)(-), CD(8)(+) was found in peripheral blood and pleural fluid, in which the clonal T cell accounted for 5.43% and 1.66% of the total lymphocytes respectively. The patient was refractory to treatment with hydroxyurea, prednisone and interferon alpha. She had poor response to a combination of therapy with low dose cytosine arabinoside, mitoxantrone, vincristine, cyclophosphamide, methotrexate and prednisone. She did not respond to imatinib and died of multiple organ failure. CONCLUSION: The present case fulfilled the WHO diagnostic criteria of FIP1L1/PDGFRalpha(-) CEL which did not respond to routine treatment and imatinib. Allogenic stem cell transplantation should be considered as early as possible in this case. It is noteworthy that clonal CD(3)(-), CD(4)(-), CD(8)(+)T-cell abnormality is related to the pathogenesis of CEL.

Detecting PML-RARalpha transcript in acute promyelocytic leukemia using real-time quantitative RT-PCR.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Establishment of a flow cytometric assay for determination of human platelet glycoprotein VI based on a mouse polyclonal antibody.

Glycoprotein VI (GPVI) is the major signaling receptor for collagen on platelets. Recent studies suggest that the surface density of GPVI is related to the activation of platelets by collagen. To measure the level of GPVI on platelets, a mouse polyclonal antibody BJ010 was prepared using an amplified fragment of extracellular domain in GPVI. The specific reactivity of BJ010 was identified by anti-GPVI specific monoclonal antibody 11a12 using immunoprecipitation, sandwich enzyme-linked immunosorbent assay (ELISA) and flow cytometry. The antibody BJ010 recognized both native and denatured human GPVI so that it was used to set up a flow cytometric assay to detect the level of GPVI in normal subjects. The relative level of GPVI on platelets was detected in 101 healthydonors. The median geometric mean fluorescence intensity (GMFI) of platelet GPVI level was 57.7. The variation between minimum and maximum values of platelet GPVI and integrin alpha2beta1 were found to be 3.5- and 4.1-fold, respectively, in the normal subjects. There was a week correlation between the amount of GPVI and integrin alpha2beta1 on platelet surfaces. For the method, the intraassay and interassay coefficient of variation was 6.3% and 8.8%, respectively. The flow cytometric assay described here provides a simple, reliable, reproducible, and readily available means of quantitation of collagen receptor GPVI density on the platelet surface in a larger number of blood samples.

[Effect of the new human transcription factor hBKLF on the proliferation, differentiation of K562 cell line and hemoglobin synthesis].

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.

[The C46359T polymorphism of DNMT3B promoter gene and pathogenesis of acute leukemia].

OBJECTIVE: To explore the relationship between the polymorphism of C46359T in DNMT3B promoter and the pathogenesis of acute leukemia (AL). METHODS: PCR-RFLP and DNA sequencing were used to analyze the genotypic polymorphism C46359T of promoter in genomic DNA of bone marrow cells/blood lymphocytes from 160 patients with AL and 240 normal controls. RESULTS: In people of the Hans in China, genotypic frequencies of 2.5% (CT), 97.5% (TT) and 0 (CC) were statistically significant (P < 0.001) comparing with the genotype frequencies of 41.8% (CT), 23.2% (TT) and 35.0% (CC) in Caucasian in USA. The genotypic frequency of CT heterozygote in 160 AL patients was 10.6%, significantly higher than that in the control subjects (2.5%, P < 0.001), indicating that the CT heterozygote might be a more frequent phenomenon in AL. Compared with TT homozygote, CT heterozygote had a 4.669-fold increased risk of acute leukemia (OR = 4.669; 95% confidence interval 1.700-14.747). It was suggested that CT heterozygote was relative to the pathogenesis of AL. CONCLUSIONS: Different distribution of genotypes in different races, the CT heterozygote was relative to the pathogenesis of AL.

[Re-expression of p16 gene in myeloma cell line U266 by arsenic trioxide].

BACKGROUND & OBJECTIVE: DNA methylation status regulates gene expression and is associated with oncogenesis. Demethylation of DNA has been proposed as a possible new strategy for cancer prophylaxis and treatment. S-adenosylmethionine is required as methyl donor for both arsenic metabolism and DNA methylation. The present study was designed to explore the possibility and mechanism of re-expression of the silenced p16 gene in human myeloma cell line U266 cells by arsenic trioxide (As2O3). METHODS: The U266 cell line in which the p16 gene is silenced due to hypermethylation was treated with different concentration of As2O3. The PCR technique combined with HpaII and its isoschizomer MspI was used to assess p16 gene methylation status. The mRNA expression levels of p16 and DNA methyltransferases (DNMTs) genes were determined with RT-PCR technique. Western blot was performed for P16 protein expression analysis. RESULTS: (1)The characteristic hypermethylation with losing expression of p16 gene in U266 cells was confirmed. After agarose gel electrophoresis, the genomic DNA digested with MspI showed a "smear" pattern, however the HpaII digested DNA showed a strong single band. There was no PCR amplified product of p16 gene when MspI digested DNA was used as the templates, whereas a 340 bp p16 gene product was found in HpaII digested DNA sample as the undigested DNA was amplified. Also, neither p16 mRNA nor P16 protein was detectable when assessed with RT-PCR and Western blot, respectively. (2)When the genomic DNA from cells treated with (0.5-2.0) micromol/L As2O3 was digested with HpaII and then analyzed by electrophoresis, a "smear" pattern was observed. The 340 bp product could not be amplified if such digested DNA was used as PCR templates. Detectable expression of both p16 mRNA and P16 protein was found in the cells treated with 1.0 micromol/L and 2.0 micromol/L As2O3. (3)Expression levels of DNMT 3A and 3B mRNA were increased in the treated cells and were dependent on As2O3 concentration, however no significant difference was found between each two groups (P >0.05). CONCLUSION: As2O3 could induce p16 gene re-expression in human myeloma cell line U266 through DNA demethylation.

[Methylation status of multidrug resistance (mdr1) gene and its correlation with expression of mdr1 gene in patients with hematologic malignancies].

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.

[The expression of DNA methyltransferase DNMT1, 3A and 3B in acute leukemia and myelodysplastic syndrome].

OBJECTIVE: To explore the relationship between methyltransferases and the pathogenesis of acute leukemia (AL) and the leukemic transformation of myelodysplastic syndromes (MDS). METHODS: Semi-quantitative RT-PCR method was used to detect the mRNA expression level of DNMT1, 3A and 3B in bone marrow cells from 75 patients with AL or MDS. RESULTS: There was no significant difference in mRNA expression level of DNMTs between a low-risk MDS group (n = 21) and a normal group. However, increased expression level of DNMT1, 3A and 3B was found in 47.6%, 47.6% and 42.9% of the patients in the low-risk group, respectively, if the upper limit of 80% of the normal controls was considered as the critical level. In high-risk MDS (n = 13), a more proportion of the cases with increased expression level of DNMTs were found, that was 53.8%, 76.9% and 92.3% respectively, and only expression level of DNMT3B was significantly higher than that in the low-risk MDS group (P < 0.01). In the AL group (n = 41) expression level of all the three subtypes was coordinately higher than that in the MDS group (P < 0.01), companying with a more frequency of 92.7%, 97.6% and 100%. Comparing with the AML group, a significantly increased expression level of DNMT1 (P < 0.01) with the same level of DNMT 3A and 3B was interestingly observed in the ALL group. CONCLUSIONS: It is possible that up-regulated DNMTs contribute to the pathogenesis of AL and the leukemic transformation of MDS, and DNMT3B might be the most important enzyme among the three subtypes.

[cDNA cloning, subcellular localization and tissue expression of a new human Krüppel-like transcription factor: human basic Krüppel-like factor (hBKLF)].

The FLD4585 clone from the cDNA library of human fetal liver may encode a hematopoietic related transcription factor. Here we tried to clone its full-length cDNA from the 22 weeks-gestation human fetal liver and study its functional domains, genomic structure, chromosomal localization, subcellular site and expression pattern. To obtain the full-length cDNA of FLD4585 clone, 5' RACE technique was used. Bioinformatics was used to analyze its genomic structure, chromosomal localization and potential functional domains. Its subcellular localization was shown by GFP fusion technique. The expression pattern was studied by Northern blot, RT-PCR and Western blot. The results show the full-length cDNA encoded by FLD4585 clone is 1810 bp long and encodes a 345 amino acids protein with high homology to mouse BKLF (basic Krüppel-like factor). Its characteristic C-terminal three contiguous C2H2 zinc fingers place it within the family of Krüppel-like factors. Bioinformatics studies show hBKLF gene spans over 33 kb on chromosome 4p15.2-4p16.1 and contains 6 exons and 5 introns. GFP-hBKLF fusion technique showed hBKLF was present in the nuclei of COS-7 cells in a punctate pattern, whereas it was absent in the nucleoli. By Northern blot, hBKLF has two transcripts, one between 4.4 kb-7.5 kb and the other between 1.35 kb-2.4 kb. The larger transcript exists widely in human tissues. However, the smaller transcript was more restricted in blood leukocytes, liver and bone marrow. RT-PCR showed erythrocytes and granulocytes could both express hBKLF and its level increased as they matured. The expression level in fetal liver decreased as it developed towards adult liver and its hematopoietic function gradually lost. Taken together, in this paper we have successfully cloned the full length cDNA of hBKLF. Expression study suggests it may have broad functions in vivo, especially the functions in hematopoietic tissues.

[Reversal effect of berbamine on multidrug resistance of K562/A02 cells and its mechanism].

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].

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.

[Apoptosis and re-expression of p16 gene in the myeloma cell line U266 induced by synergy of histone deacetylase inhibitor and demethylating agent].

BACKGROUND & OBJECTIVE: Histone deacetylation is associated with transcriptional activation controlled by DNA methylation. It is important to investigate changes of tumor cells treated with agent through two kinds of mechanisms. This study was designed to investigate the synergic effect of histone deacetylase inhibitor, sodium phenylbutyrate(SPB), and demethylating agent, 5-Aza-2'-deoxycytidine(5-Aza-CdR), on cell growth and explore the possibility of re-expression of the hypermethylated and silenced p16 gene in the myeloma cell line U266. METHODS: The cell cycle was analyzed by flow cytometry. Apoptosis was observed by transmission electron microscopy, DNA ladder, fluorescence-activated cell sorter (FACS). The expression level of p16 was detected by RT-PCR and Western blot analysis. RESULTS: The apoptotic rates of U266 cells induced by 5-Aza-CdR(1 mumol/L), SPB(1 mmol/L) alone and combination of 5-Aza-CdR and SPB were 15.09%, 89.19%, and 85.18%, respectively. The G1 phase was arrested and sub-G1 phase(50%) was induced by combination of 5-Aza-CdR and SPB. There was no G1 phase arrested when SPB or 5-Aza-CdR was used alone. The proportion of cells in G2 phase was increased with SPB alone. SPB was not able to induce the expression of p16. The expression level of p16 was induced with 5-Aza-CdR. The expression level of both mRNA and protein of p16 was increased significantly by synergy of SPB and 5-Aza-CdR. CONCLUSIONS: p16 gene in U266 cell line could be reactivated markedly with synergy of 5-Aza-CdR and SPB with cell cycle arresting in G1 phase. Meanwhile, the cell cycle phase occurring apoptosis that induced by combination of 5-Aza-CdR with SPB is different from that induced by each alone.