Fibrinolytic activity and its mechanisms in various leukemic cell lines

AIM:To study the relation between expression of uPAR and annexinⅡ and fibrinolytic activity in various leukemic cell lines. METHODS:The plasma activity was measured under the reaction between cells of NB4, SHI-1, K562, Jurkat, Raji and plaminogen by chromogenic assay. The protein expressions of uPAR and annexinⅡin cells of NB4, SHI-1, K562, Jurkat, Raji were detected by flow cytometry method. The mRNA expressions of uPAR and annexinⅡin cells of NB4, SHI-1, K562, Jurkat, Raji were detected by RT-PCR. RESULTS:The plasma activity in SHI-1 cells and NB4 cells were higher obviously than that in Raji, K562 and Jurkat cells. The protein expression ratio of uPAR and annexinⅡ in NB4 cells were (13.15±1.61)% and (95.97±1.19)%， respectively, they were (99.00±0.26)%, (90.35±2.15)% respectively in SHI-1 cells, and they were lower in K562, Jurkat, Raji cells. The expression of annexinⅡ mRNA in NB4 cells was higher than that in SHI-1 cells, and they were undectectable in K562 and Jurkat cells. The expression of uPAR mRNA in NB4 and SHI-1 cells were higher than that in Jurkat and K562 cells. The expression of uPAR mRNA in Raji cells was undectectable. CONCLUSION:The primary hyperfibrinolysis in leucocythemia cells was observed, and relation was closely with the expression of annexinⅡ. It might be the main reason for bleeding and disseminated intravascular coagulation in patients with acute promyelocytic leukemia and acute monocytic leukemia.     

Valproate enhances imatinib-induced apoptosis and down-regulates Bcr/Abl mRNA and phosphorylated protein kinase B in chronic myeloid leukemia cell line K562

AIM：To investigate the effects of valproate and imatinib on the apoptosis of chronic myeloid leukemic cell line K562. METHODS：K562 cells were divided into 3 groups and treated with valproate, imatinib and cotreatment, respectively. Cell cycle, apoptosis, the mRNA expression of Bcr/Abl, total protein kinase B (PKB) and phosphorylated PKB (p-PKB) were analyzed. RESULTS：The apoptotic rates in valproate group, imatinib group and cotreatment group were (11.47±0.25)%, (28.43±1.70)% and (57.73±4.38)%, respectively (P<0.05). No obvious difference was observed in cell cycle between cotreatment group and monodrug group. Bcr/Abl mRNA and p-PKB in the above 3 groups were (0.00±0.00), (64.17±12.27), and (0.00±0.00) ×10 9 copies/(g total mRNA), respectively (P<005), and 0.25±0.02, 0.17±0.01 and 0.08±0.01, respectively (P<0.05). No apparent difference of PKB was found in the 3 groups. CONCLUSION：Valproate enhances imatinib-induced apoptosis and may link to the down-regulation of Bcr/Abl mRNA and p-PKB in chronic myeloid leukemic cell line K562.     

Cloning of human β thalassemic mutation β IVS II654(C→T) and establishment of its mammalian expression system

AIM: To clone human β-globin gene carrying a thalassemic mutation IVS II654(C→T) and establish a eukaryotic expression system for high-level expression of human β IVS II654 gene in mouse erythroleukaemia(MEL) cells. METHODS: The fragments of human β 654 gene isolated from the β thalassemia patients homozygous for the β 654 mutation were amplified by PCR, and cloned to plasmid pBGT51. Then, the human β LCR and β 654 gene were subcloned from plasmid pBGT51 to the stable mammalian expression vector pcDNA3. 1+ together, and the MEL cells were transfected with this vector using commercially available cationic lipid FuGENE6. The MEL cells were induced for further maturation by DMSO and the expression of human β 654 gene in the MEL cells was identified by RT-PCR. RESULTS: A mammalian expression system of human β thalassemic mutation βIVS II654(C→T) was established. CONCLUSION: The level and the reliability of expression of human β 654 gene in the MEL cells in vitro are similar to that in vivo in human body. This may be a valuable gene therapy model for human β thalassemic mutation βIVS II654(C→T).     

Effect of IL-12 on the expression of Fas/FasL and TNFα

AIM:To study the effect of IL-12 on T lymphocytes apoptosis, the expression of Fas/FasL and TNFR/TNFα. METHODS:Terminal dUTP nick end labeling(TUNEL) and Annexin V assay were used. Anti-TNFR were labeled with FITC, anti-CD95 was labeled with PE and Anti-FasL with biotin. Three kinds of T cells (HTB176,TIB152 and human normal T cells) were analysed through flow cytometry. RESULTS:At 1st hour after being treated with IL-12, the expression of FasL protein and FasL mRNA in HTB176 and TIB152 began to increase and reached peak value in 24 hours. In the normal T cells, FasL just began to increase in 1 hour and maintained stability in 6, 12 and 24 hours through the later experiment period. All three kinds of T cells displayed no change in the expression of CD95 and TNFR/TNFα under the stimulation of IL-12. CONCLUSION:Expression of such apoptosis regulating factors were different in the apoptosis of T cells induced by IL-12.     

Establishment of a method to quantitatively detect FLT3 internal tandem duplication in acute myeloid leukemia with denaturing high-performance liquid chromatography

AIM: To establish a relatively-quantitative method to detect the internal tandem duplication (ITD) mutation of Fms-like tyrosine kinase 3( FLT3 )gene in acute myeloid leukemia (AML) patients using denaturing high-performance liquid chromatography (DHPLC).METHODS: According to the fact that much more FLT3 -ITD mutations are located in exon 14, we designed the primers, and use the method of polymerase chain reaction (PCR) to specifically amplify FLT3 -ITD mutation gene in 121 cases of AML, and relatively quantified the situation of mutant allelic gene of FLT3- ITD by the method of DHPLC.The effectiveness of DHPLC was verified by the method of capillary electrophoresis (CE).The sequenced results from PCR amplified products of 121 samples were compared.RESULTS: A characteristic of elution peak was detected by DHPLC with 10.7% overall positive rate (13/121) and varied in the proportion of mutant alleles,with a single duplicated insert fragment from 21 bp to 87 bp.The median range of mutant alleles was 34.5% (11.4%-80.2%).No significant difference of the positive rates and mutation proportions between the results with DHPLC and the results with CE method was observed.The results of FLT3 -ITD mutant gene of 121 samples were consistent with the results using sequencing method.CONCLUSION: A relatively-quantitative method to analyze AML patients with FLT3 -ITD mutation by DHPLC is successfully established.     

Survival analysis on aggregate data to assess time to sero-conversion after experimental infection with Bovine Leukemia virus

Bovine Leukemia virus (BLV) is a ubiquitous retrovirus that affects mainly cattle. Knowledge of the precise moment of infection is fundamental for identification and evaluation of factors related to BLV transmission. Systematic reviews and meta-analyses provide good evidence on the effects of medical interventions. The objectives were to estimate time to sero-conversion after experimental infection using data from retrieved literature and to detect factors that may influence the length of that interval using survival analysis on pooled data.

An analysis using aggregate data from 36 studies totalling 438 observations was performed. From this, four sets were created and analysed by interval-censored accelerated failure time models (AFT) with different distributions (exponential, Weibull, log-logistic, lognormal and generalized gamma), and some variants of the Cox model (Andersen-Gill, smoothing splines) with and without a frailty effect.

The AFT gamma model fit best and the estimated median time to sero-conversion in the null model was 57 days (95% confidence interval (CI): 49; 75) using all data and 47 days (95% CI: 39; 55) when only studies using experimental inoculation were considered. Some factors were consistently associated with time to sero-conversion. These included exposure by animal-to-animal contact (resulting in a seven-fold increase in time to sero-conversion compared to direct inoculation), diagnostic method to detect sero-conversion (time to sero-conversion was 1.4 times shorter when AGID was used compared to ELISA), and transmission by insect bites (biological media) delayed sero-conversion 2.3 times compared transmission via needles or other inanimate media.

After fitting a frailty Cox model, results showed that sero-conversion in susceptible animals after infection using donors, in which presence of virus before the experiment started was confirmed, increased the hazard of sero-conversion two times in comparison with donors in which virus presence was not confirmed before start of the experiment. Inoculation with blood decreased the hazard 2.5 times in comparison with lymphocyte suspensions. Heterogeneity due to different research groups was also present. Finally, a Cox model with smoothing splines contained three variables: research group, route of inoculation and a non-linear spline for infective dose. In conclusion, it can be stated some factors that influence the time to sero-conversion were identified and quantified and that a moderate influence of research centre existed. These results may contribute to the estimation of the most probable times of infection in field conditions and in a better evaluation of control measures.     

Expression of cytokines and their receptors in leukemia cell lines and normal blood cells

AIM: To study the expression of cytokines and their receptors in leukemia cell lines and normal blood cells. METHODS: RT-PCR was used to detect expression of mRNA for cytokines in leukemia cell lines(HL-60,U937,K562,HEL,DAMI,MEG-01,HUT78 and CA) and normal blood cells, including CD34⁺ cells, megakaryocytes,platelets, peripheral mononucleates cells and granulocytes. RESULTS: ①CD34⁺ cells simultaneously expressed mRNA for IL-1(α,β),IL-3, IL-6 , G-CSF, GM-CSF and their receptors and SCFR,MPL as well. The granulocytes only expressed IL-6,IL-6R,G-CSFR,GM-CSF. Megakaryocytes and platelets only expressed IL-3R,IL-6,IL-6R,MPL.Interestingly, TGFβ₁ ,TNFα and their receptors sustained to express in normal cells.②Most leukemia cell lines were found to simultaneously express at least two or more stimulating cytokines and receptors ,while TGFβ 1 , TNFα and their receptors were expressed in all the leukemia cell lines we observed. CONCLUSIONS: ①Multi-autocrine loops exist in leukemia cells;②Imbalance of autocrine loops of positive and negative cytokines may be related to leukemia.     

小鼠白血病抑制因子真核表达载体的构建及测序分析

运用RT-PCR技术,克隆含信号肽和不含信号肽的小鼠分泌型白血病抑制因子cDNA,通过pMD18-T simple载体和pBS-T载体过渡,分别构建了真核表达载体pSecTag-mlif(sp )和pSecTag-mlif(sp-),酶切进行初步鉴定。利用Blast程序,搜索NCBI GeneBank中与构建表达载体中编码MLIF cDNA的同源序列,除在编码区216bp处碱基为G和在318bp处由G突变为T外,编码LIF基因的其余序列与已发表的完全一致。运用DNAMAN软件对翻译水平进行预测,结果发现这一突变位点并不影响蛋白的翻译。     

红花苷对白血病细胞的抑制增殖和诱导分化作用

[目的]研究红花苷对K562白血病细胞的抑制增殖和诱导分化作用。[方法]以培养的K562白血病细胞为试验对象,通过XTT试验、集落形成试验、血红蛋白联苯胺染色试验以及形态学试验,考察红花苷对白血病细胞抑制增殖和诱导分化作用。[结果]红花苷剂量依赖性地抑制了K562白血病细胞的活性及集落形成(P0.05),形态学观察及联苯胺染色试验也证实了红花苷剂量依赖性地诱导K562白血病细胞向产生血红蛋白的终末细胞分化。[结论]红花苷对K562白血病细胞具有明显的抑制增殖和诱导分化作用。