Clinical Significance and Expression of PD-1/PD-L1 and Treg Cells in JAK2 V617F Positive Myeloproliferative Neoplasms / 中山大学学报(医学科学版)

@#【Objective】To investigate the expression and clinical significance of programmed death- 1（PD- 1），programmed death-ligand 1（PD-L1）and CD4+ CD25+ Foxp3+ regulatory T cells（Treg）and CD4+/CD8+ ratios in JAK2V617F mutation positive myeloroliferative neoplasms patients（MPN）.【Methods】45 cases of JAK2 V617F positive MPN patients were selected including 17 cases of essential thrombocythemia（ET），13 cases of polycythemia vera（PV）and 15 cases of primary myelofibrosis（ET）. 30 cases of the newly diagnosed group and 15 cases of treatment group were from them. 15 cases of healthy volunteers were selected as control group. The ratio of mutant and wild type of JAK2 was detected by fluorescence quantitative polymerase chain reaction（FQ-PCR）. The expression levels of p-JAK2，PD-1 and PD- L1 in pathological tissues of bone marrow were detected by immunohistochemistry. The changes of treg cells and lymphocyte subsets in peripheral blood of MPN patients and controls were detected by flow cytometry. 【Results】 The expression levels of p-JAK2，PD-1，PD-L1，and Treg in the newly diagnosed group were significantly higher than that of treatment group and control group（P<0.05），while the expression levels of CD4 +/CD8 + ratio were significantly lower than treatment group and control group（P<0.05）. JAK2 V617F mutation burden was positive correlation with PD-1 and PD- L1，and was negative correlation with CD4 +/CD8 + ，the correlation coefficients were r=0.593，P<0.01；r=0.723，P<0.01；r=-0.771，P<0.01，respectively.【Conclusion】p-JAK2，PD-1，PD-L1，Treg，CD4+/CD8+ and JAK2 V617F were involved in the pathogenesis of myeloroliferative neoplasms.

Inhibitors of JAK2/STAT Signaling Pathway and Hematologic Malignancies-Review / 中国实验血液学杂志

Jauns kinase (JAK)/transducer and activator of transcription（STAT） pathway is a classical approach to study the rapid changes of the gene expression in specific target cells by a variety of extracellular signals. The JAK and STAT transfer cytokine receptor signaling plays a unique role in multiple cellular and molecular biological changes.The abnormal signal of JAK/STAT pathway will lead to the hematopoietic abnormalities.Studies had shown that the abnormal activation of JAK2/STAT signaling pathway are in many kinds of malignant hematological diseases, such as in acute lymphoblastic/myeloid leukemia, chronic myeloid leukemia, lymphoma, myelodysplastic syndromes, myeloprofilerative neoplasm, especially in the patients of myeloproliferative neoplasm(MPN) with JAK gene mutation(JAK2V617F), this mutation has an important value for MPN diagnosis. At present, the effect of the specific inhibitors of JAK2 has showed good perspective, which had been applied to clinic treatment and achieved remarkable curative effect. In this review, the JAK2/STAT signaling transduction, the JAK2 signal and hematologic malignancies, the kagulation of signaling pathway and the inhibitors of JAK2/STAT signaling pathway are summarized.

Role of PTEN gene in multidrug resistance reversal in leukemia cells / 第二军医大学学报

Objective: To investigate the mechanism by which wild-type PTEN gene reversing multi-drug resistance (MDR) in human leukemia K562/ADM cells resistant to adriamycin (ADM). Methods: The recombinant adenovirus containing green fluorescent protein and PTEN (Ad-PTEN-GFP) or empty vector (Ad-GFP) was transducted into K562/ADM cells resistant to ADM. The transduction efficiency was assessed by flow cytometry (FCM). Then the cells were treated with different concentrations of ADM, cytarabine (Ara-C) or arsenic trioxide(As203) 3 days after transduction. The proliferation of K562/ADM cells was examined by MTT assay, the apoptosis rate was assessed by FCM, and the IC50 of different drugs was used to calculate the drug resistance reversal fold (RF), so as to observe the effect of PTEN on reversing MDR of the 3 drugs. PTEN, NF-κB, MDR1, MDR-associated protein (MRP) and apoptosis related genes (Bcl-2, Bcl-xL, Bax) were detected by fluorescence quantitative PCR. PTEN, Akt, p-Akt and NF-κB protein levels were detected by Western blotting analysis. Results: The proliferation inhibition rate and apoptosis rate of cells in Ad-PTEN-GFP plus chemotherapeutic groups were significantly higher than those Ad-GFP plus chemotherapeutic groups at 3 days after infection (MOI: 200) (P<0. 05). PTEN transduction promoted the sensitivity of K562/ADM cells to ADM, Ara-C and As203, with the RF being 3. 80, 2. 65 and 2. 64 folds, respectively. K562/ADM cells in Ad-PTEN-GFP group had lower p-Akt and NF-κB (P65) protein levels and lower NF-κB, MDRl, Bcl-2 and Bcl-xL mRNA levels, and up-regulated Bax mRNA level compared with those in Ad-GFP group. Conclusion: Wild-type PTEN gene may reverse drug resistance via inhibiting Akt pathway and regulating its downstream signaling molecules, such as NF-κB, MDR1, Bcl-2 and Bax.

Effect of proteasome inhibitor bortezomib on proliferation, apoptosis and SHIP gene expression in K562 cells / 中国实验血液学杂志

This study was aimed to investigate the effects of proteasome inhibitor bortezomib on proliferation, apoptosis and the SHIP expression of K562 cells. K562 cells were treated with bortezomib of different concentrations. Cell proliferation was analyzed by MTT assay, cell apoptosis was detected by flow cytometry and SHIP mRNA expression was assayed by RT-PCR.The results showed that after being treated with 10, 20, 50 and 100 nmol/L bortezomib for 24 h, the inhibitory rates of K562 cells were (5.76 ± 1.47)%, (10.55 ± 1.59)%, (17.14 ± 2.05)% and (27.69 ± 3.57)% respectively, and were higher than that in control (1.30 ± 0.10); when K562 cells were treated with 20 nmol/L bortezomib for 24, 48 and 72 h, the inhibitory rates of cell proliferation were (10.55 ± 1.59)%, (16.33 ± 2.53)% and (19.78 ± 1.56)% respectively, there was statistic difference of cell proliferation rate between 24 h group and 48 h group (P < 0.05). After being treated with 10,20,50,100 nmol/L bortezomib for 24 h, the apoptotic rates of K562 cells were (12.7 ± 0.6)%, (26.9 ± 0.9)%, (32.6 ± 1.2)% and (72.5 ± 1.5)% respectively,and all higher than that in control (1.0 ± 0.5)% (P < 0.05). According to results of RT-PCR detection, the expression level of SHIP mRNA was obviously up-regulated after treatment with bortezomib, and showed statistical difference in comparison with control. It is concluded that bortezomib inhibits proliferation of K562 cells in time and concentration-dependent manner and induces apoptosis through up-regulation of SHIP gene.

Effects of beta-elemene on proliferation and apoptosis of human multiple myeloma cell RPMI-8226 / 中国实验血液学杂志

This study was aimed to investigate the effect of beta-elemene on proliferation and apoptosis of human multiple myeloma cell line RPMI-8226 and its mechanism. The effect of beta-elemene on the growth of human multiple myeloma cell line RPMI-8226 was detected by MTT. The effect of beta-elemene on the apoptosis of RPMI-8226 cells was determined by flow cytometry with Annexin-V/PI staining. The effects of beta-elemene on the expression of BCL-2, caspase-3, DR-4 and NF-kappaB P65 proteins were analyzed by Western blot. The results showed that the beta-elemene obviously inhibited the proliferation of RPMI-8226 cells in both time- and dose-dependent manners. Treatment with 10 - 80 micromol/L beta-elemene for 48 hours induced apoptosis of RPMI-8226 cells in a dose-dependent manner. The expression of caspase-3 and DR-4 proteins in RPMI-8226 cells treated with beta-elemene increased in a time-dependent manner, while expressions of BCL-2 and NF-kappaB P65 proteins decreased. It is concluded that the beta-elemene can inhibit the proliferation of RPMI-8226 cells by inducing the cell apoptosis. Activating the mitochondrial and death receptor pathways of apoptosis and inhibiting the anti-apoptosis pathway may involve in the beta-elemene-induced apoptosis.

Antioncogene PTEN-a new target for myeloma therapy / 中国实验血液学杂志

Pten gene is the first antioncogene with dual phosphatase activity discovered so far, pten gene regulates the cell cycle progress, apoptosis, metastasis and invasion of the tumor cells through negatively regulating the multiple signaling transduction pathways. Multiple myeloma (MM) is a malignant tumor occurring in terminal stage of B cell differentiation. The genetic changes are considered as the important factors in MM pathogenesis, among which the deletion of antioncogene is a critical genetic change. However, little is known about the genetic change of pten in MM. This review summarizes the research advance on pten in MM including structure of pten, mechanism of pten effect and correlation of pten with MM in order to provide some references for the investigating new gene target to treat the MM.

The role of PTEN-FAK signaling pathway in metastasis and invasive ability of leukemia cells / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the effect of the wild type phosphatase and tensin homolog deleted on chromosome 10 (PTEN), a tumor-suppressor gene on the proliferation and apoptosis of human chronic myeloid leukemia (CML) cells line (K562) in vitro and explore the influence of PTEN-FAK signaling pathway on invasion and metastasis of leukemia cells.</p><p><b>METHODS</b>The recombinant Ad-PTEN gene containing green fluorescent protein gene (Ad-PTEN-GFP) or the empty vector (Ad-GFP) was transfected into K562 cells and fresh leukemia cells from CML patients in blast crisis. The growth of K562 cells was assayed by MTT assay; the apoptosis rate was assessed by flow cytometry (FCM). PTEN and FAK mRNA levels were detected by real-time fluorescent relative- quantification reverse transcriptional PCR (FQ-PCR) and its protein levels by Western blot. The metastasis and invasive ability was examined by transwell chamber assay.</p><p><b>RESULTS</b>The growth of K562 cells was suppressed markedly when Ad-PTEN-GFP was transfected into K562 cells at the 200 multiplicity of infection (MOI). The maximum growth inhibition rate was 35.2%. Transwell results showed the number of cells entered the lower chamber in Ad-GFP group was 9.1 fold more than that in Ad-PTEN-GFP group;The ability of metastasis and invasion of fresh leukemia cells was also suppressed after transfection with Ad-PTEN-GFP. FAK and p-FAK proteins were down-regulated by 0.72 and 0.16 fold lower after transfected with Ad-PTEN-GFP compared with Ad-GFP group.</p><p><b>CONCLUSIONS</b>PTEN gene might inhibit the proliferation, metastasis and invasive ability of leukemia cells via down-regulating FAK expression.</p>

Effect of rapamycin on proliferation, apoptosis and regulation of chemokine receptor CXCR4 in RPMI8226 cells / 中国实验血液学杂志

This study was purposed to investigate the effect of rapamycin on proliferation, apoptosis, cell cycle progression and the regulation of chemokine receptor CXCR4 on RPMI8226 cells. Different concentrations of rapamycin were used to treat the multiple myeloma cell line RPMI8226 for different times. The proliferation of the cells was detected by MTT assay; the apoptosis rate and cell cycle were determined by flow cytometry (FCM); apoptosis of cells was observed by inverted microscopy; the cylin D1, CXCR4 and mTOR mRNA expressions were detected by RT-PCR or FQ-PCR after treating RPMI8226 cells with different concentrations of rapamycin. The results indicated that the rapamycin could inhibit the proliferation of RPMI8226 cells and induce their apoptosis. The cell cycle was arrested at the G(0)/G(1) phase. PCR results showed the down-regulation of mTOR, cyclin D1 and mTOR mRNA expressions after treating RPMI8226 cells with different concentrations of rapamycin for 24 hours. It is concluded that the rapamycin significantly inhibits the growth of RPMI8226 cells in a dose-and time-dependent mannes and induce cell apoptosis. Cell cycle arrests at the G(0)/G(1) phase, may be due to the down-regulation of the mTOR and cyclin D1 expressions. In additions, the down-regulation of CXCR4 mRNA expression is correlated with the reduction of adhesion between myeloma cells and stromal cells.

Construction,Identification and Expression of Recombinant Eukaryotic Vector pCAG-IRES-SHIP-GFP on Porliferation of Leukemia Cell Line K562 / 中国生物工程杂志

The aim was to construct and identify the mammalian expression vector of pCAG-IRES-SHIP-GFP and to detect whether it could express in human acute leukemia cell line K562.The cDNA fragment of SHIP obtained by RT-PCR was inserted into pCAG-IRES-GFP.The recombinant plasmid was confirmed by restriction enzyme digesiton,PCR and DNA sequecing.pCAG-IRES-SHIP-GFP was transfected into K562 cells with lipofectamine 2000.The expression of SHIP was determined by GFP fluorescence and Western blot analysis.FQ-PCR was used to quantitate SHIP mRNA.The expression of p-Akt,Akt were determined by Western blot.PI were tested by flow cytometry and MTT to verify whether exogenous SHIP could inhibit proliferation of K562 cells.The results showed that the correct constrution of the recombinant plasmid pCAG-IRES-SHIP-GFP has been shown by restriction enzyme digestion,PCR and DNA sequencing.pCAG-IRES-SHIP-GFP could express SHIP protein in K562 cells.The K562 cells viability after transfected with SHIP gene droped down.Western blot analysis showed that phospha-Akt308 and Akt473 were reduced to 38.7% and 68% respectively.It was concluded that the vector of pCAG-IRES-SHIP-GFP has been successfully constructed and it can be expressed in K562 cells.The expression of exogenous SHIP gene can lead to apoptosis of K562 cells by down-regulating the p-Akt expression.What found here might be one of the mechanisms involved in the pathogenesis of leukemia.