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The purpose of study was to investigate the ultrastructural features of leukemic megakarocyte (LMK) in patients with acute megakaryocytic leukemia (M(7)). Analyzing the ultrastructure characteristics of LMK and positive ratio of platelet peroxides (PPO) in 11 patients with M(7) were analyzed on basis of transmission electron microscopic observation retrospectively. The results showed that the diameter of LMK in 7 out of 11 cases was less than 20 microm, in 2 cases of them, the LMK diameter was from 10 to 15 microm and their PPO positive ratio was more than 50%, most LMK displayed regular shape, less protrusions, irregular nucleus, high nuclear/cytoplasm ratio, tiny granules, undeveloped demarcation membrane system (DMS) and irregular tubules in cytoplasm; in 5 out of those 7 cases the diameter of LMK was about 20 microm, PPO positive cell count was from 8% to 22%, most showing round or horseshoe nuclei, more or less heterochromatin, no DMS and granules were found in LMK in 3 cases and 2 cases occasionally. In other 5 out of 11 cases, the diameter of LMK was from 20 to 40 microm and PPO positive ratio was from 16% to 80%, in which smaller LMKs were similar to those in former cases in shape, and the larger LMK had irregular protrusions, varied nuclear/cytoplasm ratio, more heterochromatin, prominent nucleolus, some of them contained developed DMS, tubules and alpha-granules. It is concluded that most patients with M(7) are predominant of LMK in stage-I and minority contained LMK in II or III stage simultaneously. The differentiation degrees of LMK are different in individual and various cases.

To investigate the cytogenetic and prognostic significance of acute myeloid leukemia (AML) with t (8; 21).189 patients with t (8; 21) AML were categorized according to their additional karyotypic aberration and their clinical outcomes analysed.Among them, 63 patients (33.3%) were t (8; 21) without other additional aberrations, 126 cases (66.7%) were t (8; 21) with other additional aberrations. -Y was found in 46.7% (63/135) of the male and -X was found in 25.9% (14/54) of female patients. In additional aberrations, loss of the sex chromosome were found in 77 cases (61.1%), Del (9q) was found in 16 cases (12.7%), +4 was found in 5 cases (4.0%); 7q- was found in 6 cases (4.8%); Tetraploidy (4N) was found in 2 cases (1.6%); Variant translocation was found in 7 cases (5.6%). The 189 patients had a high remission rate (87.0%) and a relatively long median survival (21.6 months). +4 and 4N were an unfavorable prognostic factors. Fluorescence in situ hybridization technique is a more sensitive and accurate method to detect t (8; 21), especially in variant translocation, complex variant translocation and masked translocation.t (8; 21) AML is also frequently associated with additional chromosome aberrations, these aberration had influence on prognosis.

In order to investigate the ultrastructural features of malignant T cell (MTC) in bona marrow aspirate (BMA) from patients with T Cell Lymphoma, the antigen expression of MTC was analyzed by flow cytometry, and the ultrastructural features of MTC in BMA from 13 T-cell lymphoma patients with bone marrow involvement (BMI) were observed by transmission electron microscopy. The results indicated that the sizes of MTC were uneven in every patient and their diameter were between 12 and 28 microm, in 6 out of 13 cases sizes of MTC were slightly uneven but in 7/13 cases sizes of MTC were significantly uneven. The heterochromatin of MTC was less than that of normal T cell and nucleolus diameter was from 2 to 8 microm in all cases. The nuclear contour of MTC was strikingly irregular in 10 out of 13 cases. The MTC had plenty of cytoplasm in 8 out of 13 cases and displayed many microvilli or processes on MTC surface in 7 out of 13 cases, while MTC in 6 out of 13 cases contained more Golgi's apparatuses, secretary vacuoles, dense granules and intermediate filaments. In 8 out of 13 cases mitochondria apparently swelled. It is concluded that the size of MTC increase unevenly in all patients. MTC nuclear contour in most cases is irregular by folding, indenting, and twisting, which often correlated with arising of paranuclear intermediate filaments. Processes and microvilli on surface and Golgi's apparatus, secretary vesicles, dense granules as well as intermediate filament in cytoplasm of MTC develop synchronously, meanwhile, mitochondria of MTC strikingly swell in most cases.

To elucidate effects of histone deacetylase inhibitors on cell cycle of leukemia cell lines and investigate its molecular mechanisms.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.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.PB could inhibit cell cycle of leukemia cell lines. The effects were mainly through up-regulation of p21WAF1/CIP1 expression.

To investigate the clinical and laboratory characteristics of a complex translocation t (6; 21; 8) (p22; q22; q22) in two patients with acute myeloid leukemia.Bone marrow (BM) samples were collected at presentation, prepared by short-term (24 hours) unstimulated culture and R-binding, for conventional cytogenetic assay (CCA). The complex translocation was assayed by fluorescence in situ hybridization (FISH) with a dual-color AML1/ETO-specific probe. AML1/ETO chimeric transcript was detected by reverse transcription polymerase chain reaction (RT-PCR).In both cases CCA demonstrated a complex translocation, t (6; 8; 21) (p22; q22; q22), which was confirmed by interphase and metaphase FISH and AML1/ETO fusion transcript was detected by RT-PCR. Both the two patients were diagnosed as AML-M(2), but with different immunophenotype and therapeutic outcome.The t (6; 21; 8) (p22; q22; q22) is a rare variant of complex translocation of t (8; 21) (q22; q22). More such cases are needed for elucidating its clinical features and prognosis.

To investigate the utilities of dual-color fluorescence in situ hybridization (FISH) in diagnosis and monitor of treatment in acute myeloid leukemia (AML) with t (8; 21).Seventy patients having FISH results were divided into two groups: untreated and treated group. Comparative analysis was performed between the results of conventional cytogenetic analysis (CCA) and FISH analysis, and in some of them, between FISH and reverse transcriptase polymerase chain reaction (RT-PCR) results. A successive FISH following R-banding was carried out in those with cytogenetic undetermined cases.In untreated group, 30/42 cases of t (8; 21) AML were positive for AML1/ETO in FISH assay. Three cases were positive for AML/ETO by FISH although two of them lacked t (8; 21) by CCA and one negative for AML1/ETO by RT-PCR. Six cases with complex karyotype abnormalities were confirmed to be AML1/ETO positive by the successive R-banding and FISH assay, and the involved genes were clearly visualized in FISH image. In the treated group, there were 28 cases of t (8; 21) AML diagnosed. Three cases without t (8; 21) by CCA were positive by FISH. Two patients were detected relapse earlier by FISH.The dual-color FISH technique is a much more sensitive and accurate approach to the diagnosis of t (8; 21) AML and minimal residual disease (MRD) monitoring. It can also provide precise mapping of fusion signals in complex karyotype.

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.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.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.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.

To observe the effects of AML1A and AML1B, two splicing isoforms of AML1, on the transactivation of macrophage colony-stimulating factor receptor (M-CSF-R), and explore the mechanism of hematopoietic stem cell committed differentiation and leukemogenesis.The expressive plasmids of AML1A and AML1B were constructed, and co-transfected into CV-1 cells with a luciferase reporter plasmid containing M-CSF-R promoter. The transactivity of M-CSF-R promoter was assayed by luminometer.AML1B exhibited a distinct transactivity to M-CSF-R promoter with a sequence-specificity and dosage-dependent manner. AML1A showed no any transactivity but antagonized the effect of AML1B, causing marked reduction of M-CSF-R expression.An intact structure of AML1 is necessary for transactivation of M-CSF-R. AML1A may interfere with the transactivation of AML1B, and play a key role in the fine regulation of committed differentiation of hematopoietic cell.

To explore the effect of Imatinib mesylate on proliferation, differentiation and apoptosis of leukemic Kasumi-1 cells bearing c-kit mutation.Kasumi-1 cells were treated with Imatinib at different concentrations in culture. Cell proliferation was assayed by MTT assay, expressions of c-kit antigen, surface myeloid antigen and cell cycle by flow cytometry, cell apoptosis by annexin V staining and agarose gel electrophoresis. Western blot was used to analyze the level of c-kit protein tyrosine phosphorylation.Imatinib treatment caused a time- and dose-dependent inhibition of the cell proliferation, with a 72 h IC50 of 4.45 micromol/L. Imatinib treatment induced a decrease in the mean fluorescence value of c-kit antigen, a progressive decline in S-phase cell fraction and an increase in G0/G1 cells. Treatment with 5.00 micromol/L of imatinib for 72 h induced an increase in expression of myeloid surface protein CD11, CD13 and CD15, and for 24 h induced an increase in early apoptosis cells [from 9.04% to 86.84% (P0.05)]. The apoptosis ladder was observed on agarose gel electrophoresis on 5-day treatment. Tyrosine phosphorylation level of c-kit protein was decreased by Imatinib treatment.Tyrosine kinase inhibitor Imatinib mesylate treatment could inhibit proliferation of Kasumi-1 cells which bear a c-kit mutation, induce differentiation, apoptosis and G0/G1 cells accumulation.

To explore PTEN gene expression and its clinical significance in acute leukemia.The expression levels of PTEN mRNA in 5 leukemia cell lines, 87 patients with acute leukemias (AL), including 59 acute myeloid leukemia (AML), 26 acute lymphoblastic leukemia (ALL), and 2 acute hybrid leukemia, 21 AL in complete remission (AL-CR), 31 chronic myelogenous leukemia (CML) and 14 normal controls were assayed by RT-PCR.PTEN mRNA was detected in K562 cell line, but not in Kasumi-1, HL-60, U937, Nalm-6 cell lines. The expression ratio of PTEN mRNA between CML (61.29%) and normal control (78.57%) had no statistical difference (P0.05). The expression ratios of PTEN mRNA in AL (18.39%) and AL-CR (42.86%) were significantly lower than that in normal control (P0.01 and P0.05, respectively), AL also has a lower expression ratio than that of AL-CR (P0.05). The decreased level of PTEN mRNA had a positive correlation with poor-prognostic factors (high white blood cell count ofor = 20 x 10(9)/L and chromosome abnormality).There is down-regulated expression of PTEN gene in AL. PTEN gene may play a role in leukemogenesis.

The genes encoding for the light and heavy chain variable regions were cloned by RT-PCR from a murine monoclonal hybridoma cell line, which could produce monoclonal antibody to recognize CD19 antigen on human B lymphocyte. Then fused the light and heavy chain variable regions together by a short peptide linker containing 15 amino acid (Gly4Ser)3 using splice-overlap extensive PCR. The recombinant anti-CD19- ScFv was subcloned into the expression vector pET28a and induced to be expressed by IPTG in E. coli BL21. SDS-PAGE and Western blot analysis showed that the recombinant anti-CD19-ScFv gene was expressed in E. coli BL21. ScFv expression was in the form of an inclusion bodies and the purified fusion protein was obtained after a series of purification steps including cell break, inclusion body solubilization, Ni2+ metal affinity chromatography and protein refolding. Flow cytometry analysis showed that the ScFv can react with human CD19 antigen. In conclusion, recombinant anti-CD19-ScFv gene has been successful constructed and expressed in E. coli BL21, which could provide a basic study for the future target therapy to the B lymphoid leukemia and B lymphoma.

The objective of this study was to investigate the ultrastructural characteristics of hybrid acute leukemia (HAL). Fifteen cases of HAL were studied by transmission electron microscopy (TEM), focusing on organelles and myeloperoxidase (MPO) reaction of leukemic cells. By TEM, 5 out 15 cases of HAL were consistent with immunophenotyping (3 cases of biphenotypic type, and 2 cases of biclonal type with granulocytes and lymphocytes); 2 cases were suspected as HAL. On other hand, 5 cases of HAL were assigned to ALL, and 2 cases were misinterpreted as M5a and 1 as M4b. Most of the blast cells of biphenotypic HAL showed lymphoid features, except some cases containing MPO positive granules in blasts, while a few cases exhibited monocytic or nonspecific features. TEM offers advantages in the diagnosis of biclonal type HAL and biphenotypic HAL positive for MPO. However, it is difficult to differentiate MPO-negative cases of biphenotypic HAL from ALL and a few cases may be misinterpreted as M5 by TEM.

To evaluate the prevalence of a novel FLT3 activating mutation in tyrosine kinase domain (TDK) in acute leukemia patients and its clinical implication.Genomic DNA from bone marrow mononuclear cells of 143 cases of acute myeloid leukemia (AML), 25 acute lymphocytic leukemia (ALL), 2 acute hybrid leukemia (AHL), 17 myelodysplastic syndromes (MDS) and 7 chronic myelogenous leukemia in blast crisis (CML-BC) was screened by polymerase chain reaction (PCR) and gel electrophoresis for FLT3-TKD point mutations.Among AML patients, FLT3-TKD point mutation (FLT3-TKD(+)) rate was 6.3% (9/143), an incidence significantly lower than that of internal tandem duplication (ITD) mutation (37/143, 25.9%, P0.01). According to the FAB classification, FLT3-TKD point mutation was found in 3/53 of M(2), 3/40 of M(3), 2/23 of M(5) and 1/2 of M(6) subtypes. Two patients showed both ITD and TKD mutations. No FLT3-TDK mutation was found in 25 ALL, 2 AHL, 17 MDS and 7 CML-BC patients. Sequence analysis showed that one of the mutations occurred at codon 835 of FLT3. The substitution of the first nucleotide T for G of D835, resulted in D835Y, a missense mutation. The presence of TKD mutations was related neither to age, sex, nor to WBC counts, the marrow blast percentages, and CR rates for induction therapy. CONCLUSION The incidence of FLT3-TKD(+) is significantly lower than that of ITD mutation. FLT3-TKD mutations may occur alone or together with ITD mutation. In contrast to FLT3-ITD mutation, TKD mutations were not associated with leukocytosis or low complete remission rate.

To analyse the outcome of different regimens for the treatment of patients with multiple myeloma (MM).Response rate, median survival time and overall survival rate of 206 MM patients treated with different protocols were retrospectively analysed.The median survival time, 3- and 5-year overall survival (OS) of 200 MM patients treated with conventional therapy were 30.5 months, 32.0% and 15.8%, respectively. The total response rate and complete response (CR) rate of 195 patients treated with MP regimen and combination chemotherapy (CCT) were 45.6% and 14.9%, respectively. The response rates were higher for the patients treated with CCT than for those treated with MP (50.3% versus 30.4%, P0.05). The median survival time, 3- and 5- year OS in MP versus CCT group were 30.0 versus 30.5 months, 22.0% versus 35.0%, 13.2% versus 16.7%, respectively, but all of them have no statistical difference. Compared with those without IFN alpha maintenance therapy, patients received IFN alpha therapy showed a higher response rate (34.4% versus 53.6%, P0.05) and a longer median survival time (27 versus 52 months, P0.01). The total response in patients received thalidomide was 65.5%. Of the 6 patients received hematopoietic stem cell transplantation (HSCT), 5 remained alive in CR or PR with a mean survival time of (73.0 +/- 12.5) months.CCT yields higher response rates, but not longer survival time than MP does for the treatment of MM. The response rate as well as the overall survival rate increased when IFN alpha was used as maintenance therapy. Thalidomide can improve response rate as well. HSCT could prolong survival time in patients aged60 years with good status.

To investigate whether phenylbutyrate (PB) combined with 5-aza-2'-deoxycytidine (5-Aza-CdR)could inhibit transcription repression and induce t(8;21) acute myelogenous leukemia (AML) Kasumi-1 cells to differentiate and undergo apoptosis.Kasumi-1 cells were treated with PB and 5-Aza-CdR at different concentrations in suspension culture. Cellular proliferation was determined by the MTT assay, expression of myeloid-specific differentiation antigen and cell cycles were analyzed by flow cytometry. Cell apoptosis were assessed using AnnexinV/PI staining and flow cytometry.Treatment of Kasumi-1 cells with PB caused a dose-dependent inhibition of proliferation, with an IC(50) of 2.3 mmol/L. When combined with 5-Aza-CdR, PB resulted in a greater growth inhibition with an IC(50) of 1.95 mmol/L. Treatment of Kasumi-1 cells with PB resulted in cell cycle arrest at G(0)/G(1), while combined treatment with PB and 5-Aza-CdR led to cell cycle arrest at G(2)/M. Expression of myeloid cell differentiation antigens CD11b and CD13 induced by PB was enhanced when Kasumi-1 cells were pretreated with low dose of 5-Aza-CdR. High, but not low, concentrations of 5-Aza-CdR could enhance early apoptosis of Kasumi-1 cells induced by PB.Phenylbuty rate, when combined with 5-Aza-CdR, inhibits AML cell in vitro proliferation and increases apoptosis in a synergistic fashion.

To investigate the tumor suppression efficacy of histone deacetylase inhibitor, phenylbutyrate (PB), in combination with DNA methylation inhibitor 5-Aza-2-deoxycytidine (5-Aza-CdR) in the treatment of Kasumi-1 xenograft tumor in nude mice and its mechanism.The nude mice model of Kasumi-1 xenograft tumor was established by subcutaneous inoculation. Latency of tumor formation, the ability of Kasumi-1 cells pre treated with PB to form the xenograft tumor, and the tumor suppression activity of PB and 5-Aza-CdR by intraperitoneal injection in xenografted mice model were detected. Cell differentiation and cell cycle parameters of the tumor cells were analyzed by flow cytometry analysis, apoptosis by TUNEL in situ hybridization, and tumor microvessel density (MVD) by immunohistochemistry study.The latency of tumor formation in mice with or without previous lienectomy was 17 approximately 23 and 40 approximately 50 days, respectively. Tumor cells xenografted could not be found in other tissues than in inoculation area, and still harbored the specific t(8;21) and AML1-ETO fusion gene. When the xenografted mice models treated with PB, 5-Aza-CdR, or both, the tumor growth inhibition rates were 49.07%, 25.69% and 87.46% (P0.05), the apoptosis indexes (AI) of tumor cells were (2.25 +/- 0.85)%, (1.32 +/- 0.68)%, and (5.41 +/- 1.56)% (P0.05), and the microvessel densities (MVD) were 21.69 +/- 6.25, 28.34 +/- 4.24 and 9.48 +/- 3.21 (P0.01), respectively. All the data above were significantly different from that in control (P0.05). The expression of CD11b and CD13 antigen of the tumor cells was increased in xenografted mice model treated with PB when compared with the control \[(12.08 +/- 1.02)% and (54.91 +/- 2.72)%\], respectively (P0.01), and tumor cells showed a cell cycle arrest with increased G(0)/G(1)-phase cells and decreased S-phase cells.PB inhibited the growth of Kasumi-1 xenograft tumor by inducing tumor cell apoptosis and differentiation, and suppressing its angiogenesis in vivo. 5-Aza-CdR could significantly enhance the antitumor activity of PB.

To investigate factors associated with survival of patients with Ph chromosome positive adult acute lymphoblastic leukemia (aALL) in a period of 11 years.All the clinical parameters of 31 Ph positive patients were statistically analyzed by SPSS software.Ph(+) patients account for 15.3% (31/203) of all the aALL patients. Clinically, these patients manifested older in age, higher white blood cell counts with high blast fractions and lower platelet counts (PC). Phenotypically 82.6% of them were common ALL, 39.1% coexpressed myeloid antigens, and 56.5% expressed CD34 antigen. 65.4% of them (17/26) achieved complete remission (CR) and the median remission and survival durations were 4 months and 8 months, respectively. Patients with Ph(+) and additional chromosomal aberrations accounted for 42% of all the Ph(+) patients, including monosomy 7, +Ph, del(9)(p11-12) and add/t(16)(p13), and they had lower PC as compared with those with sole Ph(+) (P = 0.012) and variant Ph translocation (P = 0.01). CD34 positive patients had a shorter remission and survival duration than CD34 negative ones (0 vs 9 months for median remission time, P = 0.024; and 6 vs 12 months for median survival time, P = 0.034). There was no evidence to support the correlation between myeloid antigen expression and survival time in these patients.Ph(+) aALL is associated with adverse prognosis and CD34 expression is a poorer prognostic factor in Ph(+) aALL patients. There is no significant clinical difference between Ph(+) aALL with or without additional chromosomal aberrations.

To evaluate the prevalence of FLT3 mutation-internal tandem duplication in acute leukemia (AL) patients and its significance.Genomic DNAs from 194 cases of AL were screened by polymerase chain reaction (PCR) and gel electrophoresis for FLT3-ITD mutations.FLT3-ITDs were found in 37 (25.9%) of 143 de novo acute myeoloid leukemia (AML) patients, including 10/53 of M(2), 15/40 of M(3), 4/20 of M(4) and 8/23 of M(5). Significantly more FLT3 aberrations were found in AML M(3) and M(5) (P0.05). No FLT3-ITD was found in 25 acute lymphoid leukemia (ALL), 2 acute hybrid leukemia, 17 myelodysplastic syndromes and 7 chronic myelogenous leukemia in blast crisis. Sequence analysis of 5 cases with abnormal PCR electrophoretic patterns revealed that the ITDs were located within exon 14 from 21 bp to 60 bp, in 3 cases the ITD was a simple tandem duplication, and in 2 cases the ITD was tandem duplication with insertion, but all of the above ITD did not altered the FLT3 reading frame. FLT3-ITD was associated with a higher peripheral white cell count (P0.05) and a lower complete remission rate (P0.05), and was more prevalent in patients with normal karyotype (P0.05).FLT3-ITD mutation occurs with a significant percentage in AML M(3) and M(5) patients. Sequences of the mutants are in frame mutation. FLT3-ITD mutation was associated with a higher peripheral white cell count and a lower complete remission rate.

To study the molecular mechanism underlying multidrug resistance (MDR) and identify unknown genes that might be involved in drug resistance development in K562/A02 cells.K562/A02 was induced by gradually increasing the ADM concentration in culture medium of K562 cells, the differential expression of associated genes between K562 and K562/A02 was determined with cDNA microarray. Overexpression of neurofilament protein NF-H gene in K562/A02 cells was confirmed with RT-PCR and immunocytochemistry. Anti-sense oligodeoxynucleotides were transfected into K562/A02 cells by lipofectamine in order to further analyze the role of NF-H in drug resistance.Comparing with the expression profiles, we found upregulation of 5 transcripts and downregulation of 7 transcripts in response to MDR of K562/A02 cells. The overexpression of NF-H, one of the 5 upregulated genes, was confirmed. After being treated with antisense oligodeoxynucleotides of NF-H and mdr1, the cellular adriamycin concentration increased significantly, but antisense NF-H alone did not have significant effect on drug resistance phenotype.The development of MDR in K562/A02 cells is multifactorial. NF-H may be involved in the drug resistance of K562/A02, which may provide a new marker of diagnosis and a new target of therapy.

To explore whether PML/RAR alpha fusion gene presented in patients with typical clinical characteristics of acute promyelocytic leukemia (APL) but normal karyotype or atypical translocation of chromosomes 15 and 17 by conventional cytogenetic analysis (CCA), and to assess the application of fluorescence in situ hybridization (FISH) to diagnosis of APL.193 newly diagnosed APL patients received CCA in our hospital, 32 cases of whom were carried out FISH analysis, and some of the patients received reverse transcriptase polymerase chain reaction (RT-PCR) detection.132 of 193 (68.4%) cases were identified to have t(15;17) (q22;q12) by CCA. The selected 32 patients were divided into three groups according to CAA results: group 1 included 14 cases with typical t(15;17), group 2 included 13 cases without t(15;17), and group 3 included five cases with complex karyotype involving chromosomes 15 and 17. As expected, all cases in group 1 were detected PML/RAR alpha fusion by FISH. In group 2, all patients presented the same molecular abnormality by FISH in spite of absence of t(15;17), and in group 3, FISH not only detected PML/RAR alpha fusion but also identified the fusion signals located on chromosomes, other than chromosome 15q.All the APL with typical clinical characteristics can be detected PML/RAR alpha fusion by FISH or RT-PCR regardless of classical t(15;17). FISH is more sensitive for molecularly diagnosis of APL, and can identify the precise location of the fusion signals in complex karyotype. It is necessary in clinically APL patients with no or atypical chromosomal abnormalities to perform FISH analysis.

To discover BCR-ABL tyrosine kinase inhibitors through structure based virtual screening.Docking screening against the distinctive inactive conformation of the catalytic domain of BCR-ABL tyrosine kinase was performed on 3D database. The MTT assay was performed to assess the viability of the tumor cells treated with selected compounds. The amount and kinase activity of BCR-ABL protein were detected in the presence of compounds by Western blot analysis and immunoprecipitation.From the top 1,000 compounds with the best DOCK energy score, 15 compounds were selected for biological assay. Eight out of 15 compounds showed notable inhibitory activity against Ph+ human K562 cells with IC50 values ranging from 10 to 200 micromol/L. In cell-based assays of ABL tyrosine phosphorylation, the ability of two kinds of novel, structurally diverse, lead compounds to inhibit ABL kinase activity was observed. However, no significant differences in the amount of BCR-ABL protein were noted on the ABL immunoblot in the presence of these lead compounds.Two promising lead compounds were discovered to inhibit BCR-ABL tyrosine kinase activity. Virtual screening technique has been proven to narrow down the size of screening compound libraries to the most prospective drug candidates with high success rates.

To explore the incidence, clinical characteristics and prognosis of childhood acute lymphoblastic leukemia (ALL) with t(12;21).t(12;21)/TEL-AML1 fusion gene was examined in bone marrow or peripheral blood mononuclear cells from 51 newly diagnosed childhood ALL patients by conventional cytogenetic R-banding analysis (CCA), dual colour interphase fluorescence in situ hybridization (I-FISH), and nested reverse transcriptase-polymerase chain reaction (RT-PCR).t(12; 21)/TEL-AML1 fusion gene was found in 11 cases by FISH or PCR, accounting for 21.6% and 26.9% in childhood ALLs and in non-T lineage ALL cases, respectively. The median age at diagnosis was 6.8 (2.9 to 12) years. All of the t(12;21) patients expressed non-T lineage immunophenotype, and most of them were common-ALL. High myeloid antigen coexpression was not found. In 11 CCA cases, normal karyotype was found in 7, and a dubious t(12;21) in one. TEL allele deletion was found in 8 (72.7%) of t(12;21) positive cases by FISH. By comparison, no statistic difference was found in sex, anemia, hemorrhage, organ enlargement, and initial WBC count between the positive and negative non-T lineage ALLs, but the platelet count and the frequency of IgH gene rearrangement were much lower in positive cases (P = 0.008 and 0.007, respectively). Moreover, no difference was found in overall CR rate, CR rate within 4 weeks, CR duration and relapse rate between the two groups.t(12;21) was the most common chromosomal translocation in childhood ALL, but not all of them could be detected by CCA. t(12;21) cases showed non-T cell immunotypes, most of them were CD(10)(+) ALL. TEL allele deletion was common in these cases. There was no significant difference in clinical characteristics and short term outcome between the t(12;21) and the TEL-AML1 negative cases. In our data, Chinese t(12;21) ALL showed older in age, lower BPC, lower IgH rearrangement frequency and more of normal karyotype as compared with the reports abroad.

To explore the blockade effect of phenylbutyrate (PB), a histone deacetylase inhibitor, on the in vitro biological function of AML1/ETO to reverse its transcription repression and induce Kasumi-1 cells to differentiate and apoptosis.Kasumi-1 cells were treated with PB at different concentrations in suspension culture. Cell proliferation was analysed by MTT assay, morphological changes by light and electron microscopy, expression of myeloid-specific differentiation antigen and cell cycle by flow cytometry, cell apoptosis by annexin V staining, agarose gel electrophoresis and flow cytometry.PB treatment caused a dose-dependent inhibition of the cell proliferation. The IC(50) was about 2.3 mmol/L. PB treatment led to a progressive decline in the fraction of S-phase cells and increase in G(0)/G(1) cells. PB induced a time- and dose-dependent increase in expression of myeloid cell surface protein CD(11b) and CD(13). A dose-dependent increase in early apoptosis for 2 days treatment, late apoptosis for 3 days treatment. The DNA ladder of apoptosis was observed on agarose gel electrophoresis for 5 days treatment. Morphological features of monocytoid differentiation and apoptosis were seen on Wright-Giemsa staining smears.PB treatment could inhibit proliferation of Kasumi-1 cells, induce partial differentiation, apoptosis and accumulation of cells in G(0)/G(1) phase.

To further verify the transcriptional repression domains in ETO and their relationship with histone deacetylase (HDAC).Either of the ETO two zinc fingers was mutated respectively by site-directing mutagenesis. The truncation fragments of ETO were amplified by polymerase chain reaction (PCR) and cloned into eukaryotic expression plasmid pFA-CMV. By the means of DNA transfection and analysis of the transcription derived from the promoter of reporter gene, the transcriptional regulation domains of ETO was determined.The expression plasmids carrying truncated ETO and ETO with point mutation at either zinc finger were successfully constructed. Two repression domains were found within ETO, which were located at two zinc finger motifs and 275 - 487 amino acid residues, respectively.The transcription repression by ETO was mediated by two separated domains and closely associated with HDAC, which may be used as therapeutic target for acute myeloid leukemia M(2b).

During a shellfish-borne hepatitis A outbreak in Shanghai during the first quarter of 1988, 300,000 cases were reported in two months. Using cell culture and experimental infection of marmosets, hepatitis A virus (HAV) was isolated from clams collected from the market and the sea bed during the epidemic. A dose-response curve correlating the quantity of clams consumed to the attack rate of hepatitis A was well documented. The occurrence of the epidemic was associated with a good harvest of clams in a new area, serious pollution of this area with sewage and importation of the clams in large quantities into Shanghai where most young adults were susceptible. Clams can apparently be decontaminated by using a continuous water flow. In this way, HAV titres can be reduced by 90% in one day and by 99.9% in two weeks. An attenuated live HAV vaccine which has been developed in China has been shown to be safe and immunogenic and may be used for prevention of such epidemics in the future.