Detection of Exosomal PML-RARA Fusion Gene Expression Level by Droplet Digital PCR / 中国实验血液学杂志

OBJECTIVE@#To establish a method for detecting the exosomal PML-RARA fusion gene expression by droplet digital PCR (ddPCR).@*METHODS@#By using Taqman probe-based ddPCR technique, the method that able to detect both long and short isoforms of PML-RARA fusion gene transcripts was established. RNA from PML-RARA negative cell line HL-60 as negative control was used to set the limit of blank (LOB), while the RNA from PML-RARA positive cell line NB4 and the recombinant plasmid pSG5-PML-RARA(S) were used to set the limit of detection (LOD) for long and short PML-RARA transcripts, respectively. Furtherly, the expression of exosomal PML-RARA fusion gene in NB4 cell culture supernatant and serum of patients with acute promyelocytic leukemia (APL) was analyzed by ddPCR technique.@*RESULTS@#The LOB of ddPCR assay for long and short PML-RARA transcripts were 0.0725 and 0.083 copies per microliter of PCR reaction system, respectively, while the LOD of long and short PML-RARA transcripts were 0.19 and 0.21 copies per microliter of PCR reaction system, respectively. In addition, the expression of exosomal PML-RARA fusion gene derived from both NB4 cell culture supernatant and serum of APL patients was successfully detected.@*CONCLUSION@#A ddPCR-based technique for detecting fusion gene transcripts has been established, which can be used to analyze absolute quantification in the minimal quantity of PML-RARA transcripts derived from exosomes. It suggests the possibility of this technique to non-invasively and dynamicly monitore the exosomal PML-RARA transcripts from APL patients' serum.

Determination of Trace ZnⅡ, CdⅡ and PbⅡ Metal Ions Using In-situ Bismuth-modified Boron Doped Diamond Electrode / 分析化学

Bismuth modified boron doped diamond (BDD) film electrode was employed for simultaneous determination of trace ZnⅡ,CdⅡand PbⅡby anodic stripping voltammetry.BiⅢwas simultaneously in-situ deposited on bismuth modified boron doped diamond electrode with ZnⅡ,CdⅡ and PbⅡ by pre-concentration.In the presence of BiⅢ,the sensitivity for determination of ZnⅡ,CdⅡ and PbⅡ was remarkably enhanced.Influence factors such as bismuth concentration,boron doped concentrations of BDD electrode,pH,preconcentration potential were investigated and optimized.Under the optimal conditions,the stripping peak currents increased linearly with the increasing concentration of ZnⅡ,CdⅡ and PbⅡ in the range of 10-300 μg/L.The limit of detection was 0.56 μg/L for ZnⅡ,0.32 μg/L for CdⅡand 0.75 μg/L for PbⅡ (S/N=3),respectively.The interference experiments showed that common ions had little influence on the determination except CuⅡ.In addition,the developed electrode displayed a good repeatability.The method was successfully applied to determination of ZnⅡ,CdⅡ and PbⅡ in real water samples with the standard addition recoveries of 92.0%-114.0%.

Effects of Quercetin on Chronic Myeloid Leukemia Cell Line Resistant to Imatinib and Its Mechanism / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To explore the growth inhibitory effect of quercetin on imatinib-resistant chronic myeloid leukemia cell lines and to clarify its involved mechanisms.</p><p><b>METHODS</b>The cell viability was detected by trypan blue Staining, percentage of apoptotic cells and cell cycle distribution were detected by flow cytometry, the protein expression was detected by Western blot.</p><p><b>RESULTS</b>Both inhibitory effect of proliferation and apoptosis-inducing effect were similar between the imatinib-resistant and -sensitive cell lines treated with 25 µmol/L quercetin for 24 hours and with arrest of cell cycle at G/M phase. Quercetin could not change the expression of BCR-ABL. The expression of γ-H2AX was markedly enhanced and the phosphorylation of JNK up-regulated by quercetin in both imatinib-resistant and imatinib-sensitive cell lines.</p><p><b>CONCLUSION</b>The growth of imatinib-resistant cells can be inhibited by quercetin, and the apoptosis of cells can be induced by quercetin, which may be related to cell cycle arrest in G/M. The DNA damage and up-regulation of p-JNK may be involved in these processes.</p>

Recent advances in biomechanical studies of abdominal aortic aneurysms / 医用生物力学

An abdominal aortic aneurysm (AAA) is a vascular pathology associated with localized and balloon-like dilatations of abdominal aorta. An untreated AAA may lead to an eventual rupture with a high mortality rate. In recent studies, the biomechanics of AAA has been widely used to assess the rupture risk in clinic. In this review paper, biomechanical testing methods on intraluminal thrombi and AAA are discussed, so as to fully understand biomechanical properties of intraluminal thrombi and aneurysmal tissues, as well as the influence of mechanical property changes on the AAA growth and remodeling under pathological environment. Then representative research findings on prediction of rupture risk by a series of experimental and computational biomechanical methods are reviewed, including finite element analysis on stress distributions on AAA wall, assessment of rupture risk index and judgment of rupture locations. The relevant microstructural changes caused by thrombus aging are described in detail, and the current situation of biomechanical studies on AAA and future challenges are briefly summarized.

Experimental Study on Apoptosis of Kasumi-1 Cells Induced by Sertraline and Its Molecular Mechanism / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate the biological effects of sertraline, one of psychotropic drugs, on actue myeloid leukemia cell line Kasumi-1.</p><p><b>METHODS</b>Cells were treated by different concentrations of sertraline for different times. The effects of sertraline were evaluated by cell growth, cell morphology, cell cycle distribution and markers of cell apoptosis, respectively. Western blot was used to detect the expression change of related proteins.</p><p><b>RESULTS</b>Sertraline could inhibit cell proliferation and induce apoptosis. After treatment with 15 µmol/L and 20 µmol/L sertraline for 24 h, the inhibitory rate of Kasumi-1 cell proliferation was (19.00 ± 7.37)% and (47.90 ± 11.19)%, respectively. Meanwhile, compared with the control group, the percentage of Annexin V positive cells in Kasumi-1 cells treated with sertraline for 24 h raised obviously from (9.71 ± 2.12)% to (20.54 ± 2.52)% and (45.37 ± 7.88)% (P < 0.01), respectively. The cells were arrested in G0/G1 and G2/M phase. In addition, it was found that sertraline could also down-regulate the level of translationally controlled tumor protein (TCTP) in Kasumi-1 cells.</p><p><b>CONCLUSION</b>Sertraline can significantly induce the apoptosis of Kasumi-1 cells, that probably is associated with the down-regulation of TCTP protein expression.</p>

Effect of bortezomib and low concentration cytarabine on apoptosis in U937 cell line / 中国实验血液学杂志

This study was aimed to explore the effect of bortezomib and low concentration cytarabine (Ara-C) on proliferation and apoptosis in U937 cell line and its mechanism. The proliferation and apoptosis of U937 cells treated with bortezomib (10 nmol/L) and(or) Ara-C (50 nmol/L) were observed by cell count, cell morphology, flow cytometry and Western blot. The results showed that bortezomib and Ara-C alone inhibited U937 cell proliferation. The inhibitory effect was enhanced by combination of these two drugs, the inhibitory rates of U937 cell proliferation were (55.00 ± 2.81)% and (70.02 ± 3.33)% after treatment for 24 h and 48 h, respectively. Bortezomib and Ara-C synergistically induced apoptosis and decreased mitochondrial membrane potential in U937 cells. The percentage of Rhodamin123 positive cells was (38.70 ± 1.54)%. Bortezomib and Ara-C also synergistically induced activation of caspase-9, caspase-8 and caspase-3. It is concluded that the bortezomib and low concentration Ara-C synergistically induced apoptosis in U937 cells, mainly through mitochondrial pathway, and possibly through death receptor pathway.

Mechanism of apoptosis synergistically induced by bortezomib combined with cytarabine in U937 cell line / 中国实验血液学杂志

This study was aimed to further explore the apoptosis-inducing effect of bortezomib combined with cytarabine (Ara-C) on U937 cell line. Proliferation and apoptosis in U937 cells treated with bortezomib and/or Ara-C were assessed by cell count. Cell cycle distribution and reactive oxygen species (ROS) production level were measured by using flow cytometry. Cell signaling pathway related to apoptosis was analyzed by Western blot. The results showed that 10 nmol/L bortezomib combined with 50 nmol/L Ara-C significantly inhibited U937 cell proliferation. These two drug combination synergistically induced apoptosis in U937 cells, significantly increased cellular ROS level, and up-regulated the expression of phosphorylated form of JNK and P38 and down-regulated phosphorylation of ERK. It is concluded that the apoptosis of U937 cells synergistically induced by bortezomib combined with low concentration Ara-C is possibly associated with up-regulation of phosphorylated form of JNK, P38 and down-regulation of phosphorylation of ERK induced by increase of ROS, resulting in decrease of mitochondrial potential.

A novel molecular mechanism of interferon alpha-regulated expression of retinoic acid-induced gene G / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To investigate the molecular mechanisms by which IFN-alpha regulated retinoic acid-induced gene G (RIG-G) expression.</p><p><b>METHODS</b>The expression of STAT1, p-STAT1 and RIG-G in IFN-alpha-treated NB4 cells was detected by Western blot. The roles of STAT1, STAT2 and IRF-9 in IFN-alpha-induced RIG-G expression were analyzed in STAT1-null U3A cells by cell transfection, reporter gene assay, co-immunoprecipitation and chromatin immunoprecipitaion.</p><p><b>RESULTS</b>In U3A cells, only when STAT2 and IRF-9 were co-transfected, the luciferase activities of RIG-G promoter-containing reporter gene could be highly increased about 8-fold compared with that in the control group. Moreover, in the absence of IFN-alpha, similar effects were observed in either IRF-9 co-transfected with wild type or mutant form of STAT2, whereas IFN-alpha could increase the transactivation activity of wild type STAT2 and IRF-9 by 6-fold compared with that without IFN-alpha, but had no effect on mutant STAT2. In addition, STAT2 could interact with IRF-9 and bind to the RIG-G promoter.</p><p><b>CONCLUSION</b>STAT2 may interact with IRF-9 in a STAT1-independent manner. The complex STAT2/IRF-9 is the key factor mediating the expression of RIG-G gene regulated by IFN-alpha. This is a novel signal transduction cascade for IFN which is different from the classical JAK-STAT pathway.</p>

Expression of ifi56 gene in ATRA-induced APL cell differentiation and construction of ifi56 gene eukaryotic expression plasmid / 中国实验血液学杂志

This study was purposed to investigate the expression of ifi56 gene in the ATRA-induced acute promyelocytic leukemia (APL) NB4 cell differentiation and to construct the eukaryotic expression plasmid of ifi56 gene. RT-PCR was used to detect the expression of ifi56 in NB4 cells treated with ATRA for different time. Human ifi56 cDNA was amplified by RT-PCR and cloned into pEGFP-C1 vector, then was transfected into 293T cells. The expression of the recombinant protein in 293T cells was detected by Western blot. The localization of IFI56 protein was observed by fluorescence microscopy. The results showed that the ifi56 mRNA was almost undetectable in untreated NB4 cells, but it significantly increased after ATRA treatment for 72 hours. The cDNA fragment of ifi56 was inserted into the expressing plasmid pEGFP-C1 successfully. The expression of EGFP-IFI56 fusion protein with a molecular weight about 83 kD was detected by Western blot. The EGFP-IFI56 protein was localized in cytoplasm mainly. It is concluded that the expression of ifi56 is enhanced significantly when the differentiation of APL cells was induced by ATRA. Gene ifi56 is successfully cloned into eukaryotic expression vector and the fusion protein is expressed in the cytoplasm mainly.

Regulation mechanism for rig-g gene expression induced by all-trans retinoic acid / 中国实验血液学杂志

To investigate the molecular mechanisms of all-trans retinoic acid (ATRA)-induced rig-g gene expression and to better understand the signal transduction of ATRA during acute promyelocytic leukemia (APL) cell differentiation, the luciferase reporter assay, co-immunoprecipitation and chromatin immunoprecipitation were used to clarify the basic transcriptional factors, which directly initiated the expression of rig-g gene. The results showed that the expression of STAT2, IRF-9 and IRF-1 could be upregulated by ATRA with different kinetics in NB4 cells. IRF-9 was able to interact with STAT2 to form a complex, which could bind the rig-g gene promoter and trigger the rig-g expression. IRF-1 alone could also activate the reporter gene containing rig-g gene promoter, but C/EBPalpha could strongly inhibit this transcription activity of IRF-1. It is concluded that during ATRA-induced APL cell differentiation, IRF-1 is first upregulated by ATRA, and then IRF-1 increases the protein levels of IRF-9 and STAT2 with the downregulation of C/EBPalpha. The complex of IRF-9 and STAT2 is the primary transcriptional factor for rig-g gene induction. This study will be helpful for better understanding the signal transduction networks of ATRA during the course of APL cell differentiation.

Role of the interferon-stimulated response elements I/II in expression regulation of the retinoic acid induced gene G / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To study the regulatory role of interferon-stimulated response elements (ISREs) located on the retinoic acid-induced gene G (RIG-G) promoter in RIG-G expression.</p><p><b>METHODS</b>By using point mutation technique, the authors constructed the wide type and site mutant reporter gene plasmids according to the ISRE sequence on RIG-G promoter, and detected the functional activities by luciferase reporter assay.</p><p><b>RESULTS</b>Mutation in ISRE II alone had no obvious effect on the expression of the reporter gene, whereas mutation in ISRE I dramatically inhibited the transactivity of RIG-G promoter. Mutation in both ISRE I and ISRE II resulted in complete loss of its response to the transcription factors for the reporter gene.</p><p><b>CONCLUSION</b>Both ISRE I and ISRE II on the RIG-G promoter are the binding sites for the complex of transcription factors. They are required for RIG-G expression, and ISRE I has a preferential role over ISRE II.</p>

In vitro study of the effects of CDA-II combined with cAMP on apoptosis induction in retinoic acid resistant acute promyelocytic leukemia cells / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the effects of CDA-II alone or combined with cAMP on the retinoic acid (RA)-resistant acute promyelocytic leukemia (APL) cells.</p><p><b>METHODS</b>The RA-resistant cell line NB4-R2 was used as an in vitro model and treated with CDA-II alone or in combination with cAMP. Cell apoptosis was assessed by morphology observation, distribution of cellular DNA contents and sub-G1 cell population. The level of Bcl-2 was detected by flow cytometry, DNA "ladder" was detected by agarose-electrophoresis.</p><p><b>RESULTS</b>CDA-II could induce NB4-R2 cell apoptosis through decreasing the level of cellular anti-apoptotic protein Bcl-2. cAMP could significantly enhance the role of CDA-II. Bcl-2 positive cell rates decreased to (15.1 +/- 4.8)% and (7.3 +/- 2.9)% in NB4-R2 cells treated with 1 mg/ml CDA-II plus 100 micromol/L cAMP for 48 h and 72 h, respectively. While 100 micromol/L of cAMP could decrease Bcl-2 positive NB4-R2 cells from (92.0 +/- 0.6)% to (75.3 +/- 2.0)%.</p><p><b>CONCLUSIONS</b>CDA-II combined with cAMP could exert potent apoptotic effect on RA-resistant APL cells.</p>

Effects of PML-RARalpha on cAMP-induced AML cell differentiation / 中国实验血液学杂志

To explore the molecular mechanisms of acute promyelocytic leukemia cell differentiation induced by cAMP combined with low-dose As2O3, the PR9 cell line, which was stably transfected by PML-RARa fusion gene, was used as in vitro model. The effects of PML-RARa on cAMP-induced AML cell differentiation were evaluated according to cell growth, cell morphology, cell surface antigen as well as luciferase reporter gene assay, in the cells before and after the treatment with cAMP and/or As2O3. The results showed that cAMP alone could slightly increase the expression of CD11b in the PR9 cells expressing the PML-RARa fusion protein, but could not induce these cells to differentiate. The cells presented the terminal differentiation morphology and significantly increased CD11b expression only under the treatment of cAMP combined with As2O3. In addition, PML-RARa had strong inhibitory activity on the transcription of the reporter gene containing cAMP response elements. In conclusions, the PML-RARa fusion protein could dramatically block the signaling pathway of cAMP during the AML cell differentiation.

cAMP analogue 8-CPT-cAMP inducing differentiation in the M2b subtype of acute myeloid leukemia cell line Kasumi-1 / 中国实验血液学杂志

This study was aimed to investigate the possible effects of cyclic adenosine monophosphate (cAMP) analogue 8-(4-chlorophenylthio) adenosine 3', 5'-cyclic monophosphate (8-CPT-cAMP) on the M(2b) subtype of acute myeloid leukemia (AML-M(2b)) cells. AML-M(2b) is characterized by the non-random chromosome translocation t (8; 21) (q22; q22), through which AML1 (acute myeloid leukemia 1) gene on chromosome 21 is fused with ETO (eight twenty-one) gene on chromosome 8, coding correspondent AML1-ETO fusion protein, which plays a crucial role in the leukemogenesis of AML-M(2b). The AML-M(2b) cell line Kasumi-1 cells were used as an in vitro model. The influences of 8-CPT-cAMP on the proliferation and differentiation of Kasumi-1 cells were evaluated according to cellular morphology, changes in cell surface antigen and cell cycle, as well as nitroblue-tetrazolium (NBT) assay. Meanwhile, semi-quantity RT-PCR and Western blot assay were used to detect the degradation of AML1-ETO fusion protein in Kasumi-1 cells before and after the treatment. The results showed that 8-CPT-cAMP (200 micromol/L) could significantly inhibit cell growth and induce differentiation of Kasumi-1 cells. However, it must be pointed out that 8-CPT-cAMP-induced differentiation in Kasumi-1 is not a typical terminal differentiation. Furthermore, 8-CPT-cAMP exerted little influence on the expression of AML1-ETO fusion gene and its product in Kasumi-1 cells. In conclusion, the 8-CPT-cAMP induced differentiation in Kasumi-1 cells. This results may provide experimental and theoretical basis for the breakthrough of differentiation-induced therapy extended to another leukemia.

Utilization of the stable ectopic expression cell line as a model for the investigation of RIG-G gene / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the biological function of RIG-G gene by establishing a cell line stably expressing RIG-G protein.</p><p><b>METHODS</b>Ectopic RIG-G gene was transfected into U937 cells by using Tet-off expression system. Changes before and after RIG-G expression were detected for cell growth, cell morphology, cell surface antigen and cell cycle regulating proteins.</p><p><b>RESULTS</b>RIG-G protein arrested the cells at G0/G1 phase and inhibited cell growth by increasing the cell cycle inhibitors P21 and P27. As compared to that in control group, the proportion of cells at G0/G1 phase in RIG-G-expressing cell group increased from (43.9 +/- 5.6)% to (63.9 +/- 2.3)% (P < 0.01). The rate of growth inhibition was (68.7 +/- 0.2)%. In addition, an increase in CD11C expression [(61.3 +/- 1.1)% vs. (18.0 +/- 0.4)% (P < 0.01)] and in cells with morphologic features of partial differentiation (smaller cell size, reduced nucleus-cytoplasm ratio, notched nucleus and coarse chromatin) was also observed in RIG-G-expressing cells.</p><p><b>CONCLUSIONS</b>RIG-G has potential abilities to inhibit cell proliferation and promote cell differentiation.</p>

Study on mechanisms of the expression regulation of interferon-induced gene RIG-G / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To investigate the molecular mechanisms of the expression regulation of retinoic acidinduced gene G (RIG-G) by interferon alpha (IFNalpha).</p><p><b>METHODS</b>RIG-G promoter region was analyzed by bioinformatics. The functional activities of RIG-G promoter with or without IFNalpha were detected by luciferase reporter assay and electrophoretic mobility shift assay (EMSA).</p><p><b>RESULTS</b>RIG-G promoter region contained two well-conserved IFN-stimulated response elements (ISREs). Both ISRE I and ISRE II showed their effective binding abilities with signal transducer and activator of transcription 1 (STAT1). In HT1080 cells, in contrast with the empty plasmid pXP2, pXP2-A reporter construct containing intact ISRE I and ISRE II showed a significant higher baseline expression (1741.2 +/- 517.5) which could be further enhanced up to three-four folds by IFNalpha (5338.7 +/- 1226.9, P < 0.05). However, the luciferase activity of pXP2-A as well as its IFNalpha inducibility could be abrogated in STAT1-deficient U3A cells (from 1741.2 +/- 517.5 to 406.1 +/- 103.2, P < 0.05), indicating that the STAT1 protein was a prerequisite for the activities of ISRE I and ISRE II.</p><p><b>CONCLUSION</b>ISREs present in RIG-G promoter region are molecular basis of IFNalpha induced RIG-G expression. RIG-G is a target gene directly regulated by STAT1 protein and should play a key role in IFNalpha signaling pathways.</p>

Activation of adenylate cyclase influences the sensitivity of acute promyelocytic leukemia cell lines to ATRA / 中华血液学杂志

<p><b>OBJECTIVE</b>To explore the molecular mechanism of APL cell resistance to ATRA.</p><p><b>METHODS</b>The ATRA sensitive and resistant APL cell lines, NB4 and NB4-R1, were used as in vitro models. The effects of specific inhibitors and activators of adenylate cyclase (AC) and phosphodiesterase (PDE) on ATRA-induced differentiation was evaluated by cell morphology, cell surface antigen expression and nitroblue-tetrazolium (NBT) reduction assays.</p><p><b>RESULTS</b>SQ22536, a specific antagonist of AC, could dramatically block ATRA-induced NB4 cell differentiation. When ATRA + SQ22536 group compared with ATRA group, the positivity of CD11b decreased from (95.9 +/- 2.5)% to (60.3 +/- 7.1)%, while the A(540) in NBT reduction assay decreased from 0.585 +/- 0.092 to 0.170 +/- 0.028 (P < 0.05). Forskolin, an agonist of AC, could overcome the resistance of NB4-R1 cells to ATRA. When ATRA + forskolin group compared with ATRA group, the positivity of CD11b increased from (34.3 +/- 5.3)% to (94.6 +/- 2.4)%, while the A(540) in NBT reduction assay increased from 0.110 +/- 0.028 to 0.395 +/- 0.049 (P < 0.05). In contrast, the specific antagonist and agonist of PDE, 3-isobutyl-1-methylxanthine (IBMX) and calmodulin, exerted little impact on ATRA treatment.</p><p><b>CONCLUSIONS</b>The defaults in the initiation of AC activation may contribute to the resistance to ATRA in some APL cells.</p>

In vitro study of the effects of arsenic trioxide combined with 8-CPT-cAMP on differentiation induction in retinoic acid resistant acute promyelocytic leukemia cells / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the potential effects of arsenic trioxide (As(2)O(3)) combined with 8-(4-chlorophenylthio) adenosine 3', 5'-cyclic monophosphate (8-CPT-cAMP) on the retinoic acid (RA)-resistant acute promyelocytic leukemia (APL) cells.</p><p><b>METHODS</b>The RA resistant APL cell lines NB4-R1 and NB4-R2 were used as in vitro models. The effect of As(2)O(3) and/or 8-CPT-cAMP was evaluated according to cellular morphology, cell surface antigen and nitroblue-tetrazolium (NBT) assay. Meanwhile, immunofluorescence analysis and Western blot assay were used to detect the degradation of PML-RAR alpha fusion protein and the change of several key cell cycle regulatory proteins in these cells before and after the treatment.</p><p><b>RESULTS</b>Low dose of As(2)O(3) (0.25 micromol/L) synergized with 8-CPT-cAMP (200 micromol/L) in inducing differentiation of NB4-R1 and NB4-R2 cells, while neither of these two drugs alone could induce differentiation of these cells. In addition, 8-CPT-cAMP was able to inhibit the cell growth by modulating the expression of some important cell cycle regulators and to facilitate the As(2)O(3)-mediated degradation of PML-RAR alpha fusion protein.</p><p><b>CONCLUSIONS</b>As(2)O(3) combined with 8-CPT-cAMP could induce differentiation of RA-resistant APL cells.</p>