Influence of Nucleostemin Expression Down-Regulation on PI3K/AKT/mTOR Signal Pathway in HL-60 Cells / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To explore the effect of nucleostemin(NS) RNAi on the expression of signal molecules in PI3K/AKT/mTOR pathway, a candidate of p53-independent signal pathway in the leukemia HL-60 cells.</p><p><b>METHODS</b>The expression of NS was interfered by transfection of P53-deficient HL-60 cells with the recombinant lentivirus expression vector NS-RNAi-GV248. The exression of NS and signal molecules of PI3K/AKT/mTOR pathway were detected by Western blot.</p><p><b>RESULTS</b>The fluorescence microscopy showed that the recombinant lentivirus vector NS-RNAi-GV248 transfected HL-60 cells successfully with a 80% transfection rate. Western blot showed that the expression of NS protein was inhibited obviously in HL-60 cells, and the expression levels of AKT, p-AKT, p70s6k and p-p70s6k were not statistically different(t=2.31,P>0.05;t=3.62,P>0.05;t=1.60,P>0.05;t=2.72,P>0.05) in comparison with control; the expression of GβL protein was statistically down-regnlated (t=15.01,P=0.002).</p><p><b>CONCLUSION</b>The changes of GβL protein correlats with NS knockdown. The PI3K/AKT/mTOR pathway may be one of nucleostemin p53-independent signal pathways.</p>

Effect of Down-Regulatory Nucleostemin Expression on Autophagy Activities in p53 Null HL-60 Leukemia Cells / 中国实验血液学杂志

<p><b>OBJECTIVE</b>Based on previous microarry and bioinformatic analysis results, to investigate the effect of nucleostemin(NS) expression down-regulation on autophagy activity in p53 null HL-60 leukemia cells, so as to provide evidence for studying mechanisms of p53-independent signal pathway of NS in details.</p><p><b>METHODS</b>The autophagy activity of HL-60 cells after down-regulation of NS expression was detected with acidine orange staining, Western blot and transmission electron mcrioscope technique.</p><p><b>RESULTS</b>The expression level of NS in test groups was lower than that in blank control and negative control groups after HL-60 cells were readily transinfected by lentivirus. The result of acidine orange staining showed that the number of acid vesicular organelle in test groups(22.4±0.76)% was higher than that in blank control groups(3.1±0.28)% and negative control groups(6.2±0.64)% (P<0.05). Western blot showed that the ratio of LC3II/LC3I in test groups(1.537±0.072) was higher than that in blank control and negative control groups (1.010±0.039) and (0.608±0.008). The result of transmission electron mcrioscopy also showed that the number of autophagosomes in test group(8.7±3.1) was higher than that in the blank control and negative control groups(4.2±1.2) and (2.3±0.5).</p><p><b>CONCLUSION</b>Autophagy activty can be enhanced after the level of NS was down regulated. The change indicates the signaling transductions screened by bioinformatic analysis may be one of p53-independent pathway of NS, which lays a foundation for contineously studying key points of p53-independent signal pathway of NS.</p>

Effect of Dexamethasone on Blast Composition in Patients with Myelodysplastic Syndrome and Its Diagnostic Significance / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To analyze the effect of dexamethason (Dex) on blast composition in patients with myelodysplastic syndrome (MDS) and investigate its significance in diagnosis of MDS.</p><p><b>METHODS</b>The flow cytometry (FCM) was used to detect the blast rate and the expression of its antigens in 30 cases of MDS (10 cases were treated with Dex as DX group and 20 cases were treated without Dex as control group).</p><p><b>RESULTS</b>The difference of the CD34(+) cell number detected by FCM was not statistically significant between DX group and control group (P > 0.05); The rate of BM B cell precursors (BCP CD34(+)/CD19(+)/CD10(+) cells) increased in DX group significantly, and BM CD117(+) cells in CD34(+) cells was decreased significantly as compared with control group (P < 0.001). The expression of antigens between granulocyte and monocyte was not significantly different (P > 0.05).</p><p><b>CONCLUSION</b>The dexamethasone can increase the rate of BCP significantly and decreased the rate of BM CD117(+) cells in CD34(+) cells significantly. There is significant influence on the blast composition in MDS patients after dexamethasone treatment and without significant influence on the other phenotypcs.</p>

Expression of CD71 on cell proliferation in hematologic malignancy and its correlation with Ki-67 / 中国实验血液学杂志

<p><b>OBJECTIVE</b>This study was to explore the expression of CD71, as a proliferation indicator, on cell proliferaration in hematologic malignancy and its correlation with Ki-67, so as to assess the feasibility of CD71 instead of Ki-67 for assaying cell proliferation by flow cytometry (FCM).</p><p><b>METHODS</b>(1) Compared with mature B lymphoctyes during stationary phase in peripheral blood from healthy people, the cell cycle and the expression of CD71 and Ki-67 of cell lines from patients with leukemia and lymphoma were examined, the correlation among CD71, S-phase cell fraction (SPF) and Ki-67 were analyzed; (2) Compared with mature B lymphoctyes in bone marrow from non-hematologic disease patients, the expression and correlation of CD71 and Ki-67 of all kinds of leukemic cells and myeloma cells from bone marrow were analyzed by using Ki-67/CD71/CD45/CD123, Ki-67/CD71/CD45/CD20 or Ki-67/CD71/CD45/CD138.</p><p><b>RESULTS</b>(1) in respect to the expression rate of CD71 on tumor cell lines, the expression rate of CD71 on HL-60 cells was (99.77 ± 0.064)%, the expression rate of CD71 on NB4 cells was (99.23 ± 0.12)%, the expression rate on THP-1 cells was (98.90 ± 0.30)% and the expression rate on K562 cells was (97.03 ± 0.15)% in myelogenous leukemia cell lines, the expression rate of CD71 on Raji cells was (99.35 ± 0.21)% and the expression rate on Mino cell was (96.95 ± 0.42)% in lymphoma cell lines, which were also obviously higher than that on cells of the control group (P < 0.05); (2) in respect to the expression rate of CD71 on tumor cells in bone marrow, the expression rate of CD71 on poorly differentiated AML(M1 and M2) cells was (51.50 ± 19.31)%, the expression rate of CD71 on acute promyelocytic leukemia (AML-M3) cells was (35.71 ± 14.02) %, the expression rate of CD71 on acute monocytic leukemia (AML-M5) cells was (30.54 ± 14.38)%, the expression rate of CD71 on acute T lymphoblastic leukemia cells was (68.40 ± 20.83)%, the expression rate of CD71 on acute B lymphoblastic leukemia was (39.67 ± 18.27)%, the expression rate of CD71 on multiple myeloma (MM) cells was (55.49 ± 18.15%), the expression rate of CD71 on chronic lymphocytic leukemia(CLL) was (1.32 ± 0.33%), which were also higher than that on cells in the control group(P < 0.05) except for CLL cells (P > 0.05); (3) CD71 had a positive linear corrlation with SPF in cell lines (r = 0.914, P < 0.05), and also had a positive linear corrlation with Ki-67 in cell lines and carcinoma cells from bone marrow (r = 0.894,r = 0.904, P < 0.05).</p><p><b>CONCLUSION</b>The CD71 can take the place of Ki-67 as an indicator of cell proliferation activity of hematologic malignancies and the determination CD71 by FCM is simpler and better than that of Ki-67 in respest of methodology.</p>

Down-regulatory effect of Nucleostemin expression on signal molecule of PI3K/AKT/mTOR pathway in HL-60 cells / 中国实验血液学杂志

This study was purpose to explore the down-regulatory effect of nucleostemin (NS) expression on signal molecules of PI3K/AKT/mTOR pathway belonged to candidate ways of p53-independent signal pathway in the leukemia cells. The expression of NS was interfered by using recombinant lentivirus expression vector NS-RNAi-GV248 to transfect HL-60 cells of p53 deficiency. The expression of NS and signal molecules of PI3K/AKT/mTOR pathway were detected by using Real-time PCR. The results of showed that the HL-60 cells were transfected by recombinant lentivirus vector NS-RNAi-GV248 successfully and with transfection rate up to 80%. According to results of Real-time PCR detection, the inhibition rate of NS gene was 56.5% in HL-60 cells. And the expression levels of PI3K,AKT and GβL mRNA (0.491 ± 0.084,0.398 ± 0.164, 0.472 ± 0.097 respectively) were obviously down-regulated by silencing NS, and showed statistical difference (P < 0.05) in comparison with control (1.002 ± 0.171, 1.000 ± 0.411, 1.001 ± 0.206 respectively) . It is concluded that the changes of signal molecules of PI3K/AKT/mTOR pathway positively correlate with NS down-regulation, which provides evidence for confirming PI3K/AKT/mTOR signal pathway possible as a type of NS p53-independent pathway.