Inhibitory Effect of NPM Gene Knockdown on Proliferation of Chronic Myeloid Leukemia Cell Line K562 and Its Mechanism / 中国实验血液学杂志

OBJECTIVE@#To investigate the role of nucleophosmin (NPM) in the proliferation of chronic myeloid leukemia cells (K562 cells) and its mechanism by RNAi technology.@*METHODS@#shRNA was used to inhibit the expression of NPM. The expression of NPM gene was detected by real-time quantitative PCR. The effect of inhibiting NPM gene on cell proliferation was detected by MTS assay. Change of cell cycle was detected by flow cytometry. Western blot was used to detect the expression of cell cycle-related proteins.@*RESULTS@#The shRNA lentiviral vector targeting at NPM gene was successfully constructed and used to transfect the K562 cells. The results showed that compared with the control groups, suppression of NPM gene expression in K562 cells could inhibit the cell proliferation and decrease the cell colony formation. Moreover, interference of NPM gene could prolong G/G phase and arrest cell cycle, which may be related to the down-regulation of NPM gene expression and activation of p21 protein expression, thereby inhibited the formation of CDK2/ Cyclin E complex.@*CONCLUSION@#Down-regulation of NPM gene expression in K562 cells can induce cell cycle arrest and inhibit cell proliferation.

Effect of Inhibiting NCL Expression on Drug-Resistance of Chronic Myeloid Leukemia Cell Line K562 and Its Resistant Cells / 中国实验血液学杂志

OBJECTIVE@#To construct a K562 and adriamycin-resistant K562 (KAR) cell line with stably down-regulation of NCL (nucleolin) expression, and to investigate the effect of NCL down-regulation on the drug resistance in K562 and KAR cells.@*METHODS@#K562 and KAR cells were infected with lentivirus, and stably transfected cell clones were obtained by puromycin screening. The cell proliferation was detected by MTS assay, the cell apoptosis was detected by flow cytometry, and the expression level of drug resistance related genes was detected by real-time PCR.@*RESULTS@#The K562 and KAR cells with stable down-regulation of NCL were successfully constructed. Compared with the control group, the proliferation of K562 and KAR cells with down-regulating NCL expression decreased significantly (P ＜0.05), the apoptosis of cells increased significantly (P ＜0.05), and cell resistance to adriamycin was down-regulated.@*CONCLUSION@#Inhibition of NCL expression may increase the sensitivity of cells to adriamycin, which may be related with the promotion of apoptosis of K562 and KAR cells.

Effect of Baicalin Derivative 02-036 on Burkitt Lymphoma Cell Line CA46 and Its Related Mechanisms / 中国实验血液学杂志

OBJECTIVE@#To investigate the effect of baicalin derivative 02-036 on proliferation and apoptosis human Burkitt lymphoma cell line CA46 and its related mechanisms.@*METHODS@#The MTT assay and cell colony formation assay were used to measure the growth inhibition of CA46 cells after 02-036 treatment. The flow cytometry with AnnexinV-FITC/PI double staining was employed to detect the apoptosis induction effect of 02-036 on CA46 cells. Cell cycle distribution of CA46 cells was estimeted by using DNA ploid analysis. Western blot was used to determine the changes of apoptosis-related proteins, including C-MYC, BCL-2, Procaspase-9, Procaspase-3, PARP and Cleaved-PARP.@*RESULTS@#Baicalin derivative 02-036 obviously inhibited the proliferation of CA46 cells, with dose- and time-dependent manner (r=0.963, r=0.992). The averaged IC value of CA46 cells was (6.04±0.11) μmol/L after 48-hour treatment. Low concentration of 02-036 could significantly inhibit the colony formation of CA46 cells. Flow cytometry analysis confirmed that 02-036 could effectively induce CA46 cell apoptosis. The apoptosis rate correlated with drug concentrations (r=0.959). Also, DNA ploid analysis showed that the cell cycle of CA46 was arrested in the S phase. The expression levels of BCL-2, Pro-caspase-9, Pro-caspase-3, PARP and C-MYC proteins decreased with a 02-036-dose dependent manner (r values were -0.990, -0.939, -0.971 and -0.967, respectively). In contrast, the expression level of cleaved-PARP increased with the same manner (r=0.920).@*CONCLUSION@#Baicalin derivative 02-036 can effectively inhibit the proliferation and induce apoptosis of CA46 cells, and its related mechanisms may be correlated with the down-regulation of apoptosis-related molecule expression levels, such as BCL-2, Pro-caspase-9, Pro-caspase-3, PARP and C-MYC.

Effect of A Novel Emodin Derivative on Chronic Myelogenous Leukemia K562 Cells and Imatinib-resistant K562/G01 Cells / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To explore the effect of a novel emodin derivative E19 on proliferation inhibition and apoptosis induction of human chronic myelogenous leukemia (CML) cell line K562 and imatinib-resistant CML cell line (K562/G01), and to clarify the involved mechanisms.</p><p><b>METHODS</b>MTT and colony formation test were used to detect the cell proliferation. Apoptotic induction effects were examined by DAPI staining method and DNA ladder assay. Western blot was performed to detect the changes of P210(Bcr-Abl) protein.</p><p><b>RESULTS</b>The emodin derivative E19 could efficiently inhibit proliferation and induce apoptosis in K562 and K562/G01 cells. IC50 of K562 cells and IC50 of K562/G01 cells were (1.20 ± 0.19) µmol/L and (1.22 ± 0.16) µmol/L, respectively. DNA fragmentation in K562 cells and K562/G01 cells confirmed that the E19 induced apoptosis in dose-dependent manner. Western blot showed that emodin derivative inhibited phosphorylation of P210 protein in K562 cells and K562/G01 cells and down-regulated the expression level of P210 in dose- and time-dependent manners.</p><p><b>CONCLUSION</b>The emodin derivative E19 can efficiently inhibit growth and induce apoptosis of K562 cells and K562/G01 cells, while the inhibition of phosphorylation of P210 protein and down-regulation of P210 protein expression may be involved in these processes.</p>

Emodin Induces Apoptosis of K562/Adr Cells Probably through Akt-Caspase 3 Signal Pathway / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate the apoptosis-inducing effects of emodin on multidrug resistant leukemia cell line K562/Adr, and to explore the role of Akt-Caspase 3 signal pathway in apoptosis of K562/Adr cells treated with emodin.</p><p><b>METHODS</b>K562/Adr cells were exposed to emodin of different doses. The ability of emodin to induce apoptosis of K562/Adr cells was detected by Annexin V/PI double labeled flow cytometry and DNA ploidy analysis, the expressions of procaspase-3, PARP, Akt, p-Akt protein were determined by Western blot.</p><p><b>RESULTS</b>Apoptosis in K562/Adr cells could be induced by emodin in a dose dependent manner, Western blot results showed that emodin down-regulated the expression levels of procaspase-3, Akt, p-Akt, PARA 116 KD in treated K562/Adr cells, up-regulated expressions leves of PARP 85 KD in a time-dependent manner.</p><p><b>CONCLUSION</b>The Akt-Caspase 3 signal pathway may be involved in these processes.</p>

Expression and significance of stathmin1 in acute leukemia / 中国实验血液学杂志

This study was aimed to investigate the expression of stathmin1 mRNA and stathmin1 protein in de novo patients with acute leukemia (AL), relapsed patients with AL and complete remission patients with AL, and its clinical significance. The expression of stathmin1 mRNA and stathmin1 protein in peripheral blood samples from 76 cases of AL and 25 healthy persons were examined by fluorescent quantitative PCR (FQ-PCR) and Western blot, respectively. The results showed that the stathmin1 protein expression could not be detected in healthy persons, only the low level of its mRNA could be observed in them. The stathmin1 mRNA expression level in de novo AL patients was higher than that in healthy persons (P < 0.05), the stathmin1 mRNA expression level in relapsed patients with AL was higher than that in de novo patients (P < 0.05), and there was no significant difference of stathmin1 mRNA expression between patients with AML and patients with ALL. The positive rate of stathmin1 protein expression in de novo patients with AL was 89%, while it obviously decreased or did not express in complete remission patients with AL. The stathmin1 protein expression in relapsed patients with AL did not display significant difference as compared with that in de novo patients (P > 0.05). There was no significant difference in stathmin1 protein expression between patients with AML and patients with ALL (P > 0.05). It is concluded that stathmin1 protein and mRNA are overexpressed in de novo patients and relapsed patients, and lowly expressed in complete remission patients. Therefore, the stathmin1 may be a new biological marker for evaluation of minimal residual disease.

Up-regulation of Stathmin and CrkL protein expressions in adriamycin-resistant leukemia cell line K562/A02 / 中国实验血液学杂志

The purpose of this study was to compare the differences of the protein expression profiles between human myeloid leukemia K562 cells and adriamycin-resistant K562/A02 cells, as well as to select novel resistance-related proteins in myeloid leukemia by means of proteomics. The total cellular proteins were separated from K562 and adriamycin-resistant K562/A02 cells by using technique of two dimensional difference in gel electrophoresis (2D-DIGE). Differentially expressed proteins were analyzed by matrix-assisted laser desorption ionization/time of flight-mass spectrometry (MALDI-TOF/MS), and by protein database searching. Moreover, the differentially expressed proteins were verified at protein and mRNA levels by Western blot assay and quantitative real time PCR. The results showed that 8 proteins differentially expressed in adriamycin-resistant K562/A02 cells, among them 2 proteins were identified to be down-regulated and 6 to be up-regulated. These identified proteins involved in the cell energy metabolism, cell proliferation, cell apoptosis, signal transduction, gene transcription and translation respectively. The results assayed by Western blot were similar to those detected by 2D-PAGE. Two up-regulated proteins Stathmin and CrkL were selected for verification in K562 and K562/A02 cells. As a result, the results detected by Western blot were identical with results from 2D-DIGE; real time quantitative PCR assay showed that the changes of CrkL at mRNA level were identical with changes at protein level, but no complete identity of Stathmin changes at mRNA level and protein level was observed. It is concluded that the difference of protein expression profile exists in K562 and K562/A02 cells. Stathmin and CrkL proteins may be involved in the drug resistance and suggest a novel clue for the resistant mechanisms in myeloid leukemia, which is worth further to explore.

Establishment of nucleophosmin gene silenced HL-60 and its resistant cell line / 中国实验血液学杂志

This study was aimed to construct model cell line of NPM1-RNAi in HL-60 cells and its resistant line (HL-60/ADR) so as to provide a experimental basis for investigating the potential role of NPM1 gene in leukemia drug resistance. The shRNA targeting to NPM1 was ligated into linear pGCSIL-GFP vector, and transformed into E.coli DH5α. Positive clone was identified by PCR and DNA sequencing. pHelper 1.0, pHelper 2.0 and pGCSIL-GFP-NPM1-shRNA were cotransformed into 293T cells by lentivirus vector system. NPM1-RNAi-LV was transfected into HL-60 and HL-60/ADR cell lines. The efficiency of NPM-RNAi-LV was detected by using real-time quantitative RT-PCR and Western blot. The results showed that the recombinant eukaryotic expression vector pGCSIL-GFP-NPM1-shRNA was constructed. pGCSIL-GFP-NPM1-shRNA was packed into NPM1-RNAi-LV by lentivirus vector system, and transfected into HL-60 and HL-60/ADR cell lines. At mRNA level, the efficiency of NPM1 mRNA knockdown was more than 90% (p < 0.05). At protein level, obvious down-regulation of NPM protein was noted, indicating that NPM1 gene in HL-60 and HL-60/ADR cell lines was knocked down after transfected with NPM1-RNAi-LV. The resistance of HL-60/ADR cell line to adriamycin decreased to a certain degree after NPM1 gene silencing. It is concluded that the model cell lines of NPM1-RNAi in HL-60 and HL-60/ADR are successfully constructed, which can be used for investigating the potential role of NPM1 gene in drug resistance of leukemia.

Effect of emodin on proliferation inhibition and apoptosis induction in leukemic K562 cells / 中国实验血液学杂志

The study was aimed to investigate the effects of emodin on proliferation inhibition and apoptosis induction in human chronic myeloid leukemia cell line K562 cells, and to explore the role of P210 protein and activation of caspase 3 in these processes. K562 cells were exposed to emodin at different doses. The proliferation inhibition was detected by MTT assay and colony formation test. The ability of emodin to induce apoptosis and DNA fragmentation were examined by flow cytometry. The expressions of P210, procaspase-3 and PARP protein were determined by Western blot. The results indicated that the emodin remarkably inhibited the K562 cell proliferation, with IC(50) value of 38.25 micromol/L after treatment for 48 hours. Meanwhile induced apoptosis, Annexin V-FITC positive cells, sub-G(1) apoptotic peak and DNA fragmentation in K562 cells confirmed that emodin induced apoptosis in K562 cells in dose-dependent manner. Western blot results showed that emodin inhibited phosphorylation of P210 protein in K562 cells and down-regulated the expression levels of P210. The procaspase-3 level in treated K562 cells decreased with increased expressions of PARP in time-dependent manner. It is concluded that the emodin efficiently inhibits growth and induces apoptosis of K562 cells, while the inhibition of phosphorylation of P210 protein, down-regulation of P210 protein expression and activation of caspase-3 may be involved in these processes.

Two novel splicing variants of eIF4E obtained by electronic cloning technique combined with RT-PCR / 中国实验血液学杂志

In order to clone the full-length cDNA of a novel EST which is probably related to acute leukemia relapse and to analyse the sequences, the electronic cloning technique combined with RT-PCR was used to clone the full-length cDNA, and the sequences were analyzed by bioinformatics. The results showed that the two novel splicing variants of eIF4E named as splicing variant 1 and 2 of eIF4E were obtained. Bioinformatics analysis showed that variant 1 and 2 exhibited 84% and 47% similarity to eIF4E mRNA, and were localized on eIF4E locus on chromosome 4. The lengths of 2 variants are 1 904 bp and 3 393 bp, encoding 245 amino acids and 132 amino acids respectively. BLAST results showed that the both variants mentioned above contains seven exons. Among them, the sequences of the three exons at 5' end of variant 1, variant 2 and eIF4E mRNA were different from each other. Protein BLAST showed that they are partially different from eIF4E protein. It is concluded that the two novel splicing variants of eIF4E were cloned, and their relation to acute leukemia relapse needs to be further investigated.