Humans ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; metabolism ; Proto-Oncogene Proteins c-akt ; metabolism ; Signal Transduction

OBJECTIVE: To investigate the effect of silencing DNA methyltransferase 1(DNMT1) to the methylation of the promoter of the tumor suppressor gene wnt-1 (WIF-1) in human chronic myeloid leukemia (CML) cells. METHODS: DNMT1 siRNAi plasmid was constructed and DNMT1 siRNAi was transfected into CML K562 cells. RT-PCR and Western blot were used to detect the expression of DNMT1 gene and related protein, and methylation PCR was used to detect WIF-1 gene promoter methylation level. The trypan blue exclusion and MTT assay were used to detect the cell proliferation, flow cytometry were used to detect the cell apoptosis rate, colony formation assay was used to detect cell colony formation ability. Expression of Wnt/β- catenin and its downstream signaling pathway proteins were detected by Western blot after DNMT1 gene was silenced. RESULTS: The expression level of DNMT1 mRNA and its related protein in the experimental group were significantly lower than those in the control group and negative control group (P<0.05). After 72 hours of successful transfection, the WIF-1 gene in the control group and negative control group were completely methylated, while in the experimental group, the methylation level significantly decreased. The results of MSP showed that the PCR product amplified by the unmethylated WIF-1 primer in the experimental group increased significantly,while by the methylated WIF-1 primer decreased significantly. After 48 h of transfection, the OD value, viable cell number and colony formation of the cells in experimental group were significantly lower than those in the negative control group and the control group (P<0.05). The apoptosis rate of the cells in experimental group was significantly higher than those in the negative control group and control group (P<0.05). The expression levels of β- actin, myc, cyclin D1 and TCF-1 in K562 cells in the experimental group were significantly lower than those in the negative control group and control group (P<0.05). CONCLUSION Silencing DNMT1 gene can inhibit the proliferation and promote the apoptosis of K562 cells. The mechanism may be related to reverse the hypermethylation level of the WIF-1 gene promoter, thereby inhibit the activity of the Wnt/β- catenin signaling pathway.
Adaptor Proteins, Signal Transducing/metabolism* ; DNA Methylation ; Humans ; K562 Cells ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive/genetics* ; Repressor Proteins/metabolism*

This study was aimed to investigate the bcr-abl transcription level and its relationship with the clinical status of patients so as to provide some bases for predicting patient status according to absolute value of bcr-abl transcript. The bcr-abl/abl values (%) of bone marrow samples from 30 newly diagnosed CML patients at the baseline bcr-abl/abl value obtained in CML patients with bcr-abl positive were defined, then 161 bone marrow samples from 82 patients were detected at virions time points, and the bcr-abl/abl value of each sample was compared with baseline value and its relationship with clinical status of patient at same time point was investigated. The results showed that bcr-abl/abl values (%) of 30 patients showed positive skew distribution and a large variation with mean 13.5631 (1.0206 - 98.3159) and mathematical mean of 21.1491 (95% CI: 12.3532 - 29.9450). For strict standard, the baseline value of bcr-abl/abl (%) was set as 1, the lower limit of these values. In the detected results of 161 samples, there were 33 samples' values above the baseline value, in which resistance/relapse/progression (R/R/P) 13 (39.4%, 13/33), no remission (NR) 17 (51.5%, 17/33) and complete hematologic remission (CHR) 3 (9.1%, 3/33) were observed. the values of 26 samples decreased by 0 - 1 order of magnitude (0.1 < or = bcr-abl/abl % < 1), in which R/R/P 6 (23.1%, 6/26), NR 7 (26.9%, 7/26), CHR 7 (26.9%, 7/26) and cytogenetic remission (CyR) 6 (23.1%, 6/26) were observed, the values of 19 samples decreased by 1 - 2 order of magnitude (0.01 < or = bcr-abl/abl % < 0.1), in which NR 2 (10.5%, 2/19), CHR 3 (15.8%, 3/19) and CyR 14 (73.7%, 14/19) were determined. 7 samples decreased by 2 - 3 order of magnitude (0.001 < or = bcr-abl/abl % < 0.01) in which major CyR (MCyR) 2 (28.6%, 2/7) and complete CyR (CCyR) 5 (71.4%, 5/7) were determined, the values of 76 samples decreased by 3 or more order of magnitude (bcr-abl/abl % < 0.001), and all these were CCyR. In conclusion, the using decrease degree of one time point-detected value compared to the baseline could well assess the patient clinical status. The bcr-abl/abl % < 0.01 can reliably reflect CyR obtained by patients at the time point, and bcr-abl/abl % < 0.001 can reflect CCyR obtained by patients. However, exact judgments of patient status relies on dynamic and serial monitoring.
Adolescent ; Adult ; Female ; Fusion Proteins, bcr-abl ; analysis ; metabolism ; Humans ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; metabolism ; pathology ; physiopathology ; Male ; Middle Aged ; Polymerase Chain Reaction ; methods ; Young Adult

The aim of this study was to investigate the apoptosis-inducing effect of cinnamic aldehyde (CA) on chronic myeloid leukemic (CML) cells and its mechanism. K562 cells and primary bone marrow mononuclear cells (MNC) from patients with CML were treated by various concentrations of CA. Flow cytometry was employed to measure the apoptosis of K562 cells and primary CML bone marrow MNC. Western blot was used to determine the expression of C-MYC and the phosphorylation of CrkL in K562 cells, and real-time polymerase chain reaction (real-time PCR) was used to quantify the expression of BCR-ABL mRNA in K562 cells. The results indicated that CA induced the apoptosis of K562 cells in a time- and dose-dependent manner. CA induced apoptosis of CML MNC dose-dependently. CA inhibited the expression of BCR-ABL mRNA and C-MYC, reduced CrkL phosphorylation levels in K562 cells. It is concluded that CA induces apoptosis of CML cells in vitro. Down-regulation of the expression and function of BCR-ABL may be one of its most important anti-leukemia mechanisms.
Acrolein ; analogs & derivatives ; pharmacology ; Apoptosis ; drug effects ; Fusion Proteins, bcr-abl ; metabolism ; Gene Expression Regulation, Leukemic ; Humans ; K562 Cells ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; metabolism ; pathology

OBJECTIVE: To explore the expression of CPEB4 in K562 cells, the biological activity and its possible molecular mechanisms. METHODS: Western blot was used to detect the expression of CPEB4 in normal leukocytes and K562 cells. The overexpression plasmid pcDNA3.1(+)-His-CPEB4 and the silencing plasmid pPLK+Puro-CPEB4 shRNA were transfected into K562 cells to change the expression of CPEB4 in K562 cells, and the transfection efficiency was detected by Western blot. Finally, CCK-8 and flow cytometry were used to detect the proliferation and apoptosis of differently treated cells, and the expression changes of proliferation and apoptosis marker proteins (AKT, p-AKT, caspase-3, BCL-2) were detected by Western blot. RESULTS: Compared with normal leukocytes, the expression of CPEB4 protein in K562 cells was higher (P＜0.01). Compared with the control group, the proliferation of CPEB4-silenced K562 cells significantly increased (P＜0.01), the number of apoptotic cell significantly decreased, and expression of AKT, p-AKT and BCL-2 was significantly increased, the protein expression of caspase-3 was significantly reduced. The proliferation of K562 cells after CPEB4 overexpression was slowed down (P＜0.05), the number of apoptotic cells significantly increased,the expressions of AKT, p-AKT and BCL-2 were significantly down-regulated, and the expression of caspase-3 was up-regulated. CONCLUSION CPEB4 can inhibit the proliferation and promote the apoptosis of K562 cells, the AKT, p-AKT, BCL-2 and caspase-3 are involved in the regulation mechanism.
Apoptosis ; Cell Proliferation ; Humans ; K562 Cells ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; RNA-Binding Proteins ; metabolism ; Transfection

OBJECTIVETo explore the effects of As(2)O(3) on BCR-ABL protein level and signal transduction in chronic myeloid leukemia (CML) cells.

METHODSImmunoprecipitation, protein tyrosine kinase (PTK) activity assay, real-time Taqman quantitative PCR and Western blot were used.

RESULTSAs(2)O(3) downregulated BCR-ABL protein and STAT1 protein of CML mononuclear cells in the concentrations of 1.0 approximately 2.0 micro mol/L and 0.5 approximately 2.0 micro mol/L after 48 h exposure, respectively. However, p27 protein level was not affected. The PTK activity of BCR-ABL protein was also mildly decreased in CML monouclear cells at 60 h. The bcr-abl mRNA level remained unchanged.

CONCLUSIONAs(2)O(3) downregulats BCR-ABL protein, STAT1 protein, BCR-ABL signal transduction and PTK activity in CML cells.

Fusion Proteins, bcr-abl ; genetics ; Humans ; K562 Cells ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; genetics ; Phosphorylation ; drug effects ; Protein-Tyrosine Kinases ; metabolism ; Signal Transduction

OBJECTIVETo detect bcr/abl fusion gene at DNA level in K562 cell line and mononuclear cells from patients with chronic myelogenous leukemia (CML).

METHODSBased on previous research, a set of DNA-PCR primers was redesigned. DNA from K562 cells and mononuclear cells of CML patients was extracted respectively. After DNA-PCR for bcr/abl fusion gene the amplified fragments were then sequenced.

RESULTAt DNA level the bcr/abl fusion gene in K562 cells and mononuclear cells of 2 CML patients was amplified. Furthermore, the DNA breakpoint of fusion gene in the above samples through sequencing of amplified fragments was localized.

CONCLUSIONDNA-PCR offers a new detection technology for bcr/abl fusion without the expression of fusion gene.

Fusion Proteins, bcr-abl ; genetics ; Humans ; K562 Cells ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; genetics ; Leukocytes, Mononuclear ; metabolism ; Polymerase Chain Reaction ; methods

OBJECTIVETo investigate the relationship between cyclin D2 and P210(BCR/ABL) tyrosine kinase in chronic myelogenous leukemia (CML).

METHODSRT-PCR, Western blot and flow cytometry were performed to detect the expression of cyclin D2 in K562 cells and in K562-ib-eGFP cells which express intracellular single-chain antibody (sFv, intrabody) against ABL tyrosine kinase domain.

RESULTSCyclin D2 expression in K562-ib-eGFP cells was 18.90% which was lower than that of control K562 cells (48.10%), and the number of S-phase cells in K562-ib-eGFP was 40.40% which was much lower than that in K562 cells (64.34%).

CONCLUSIONCyclin D2 is a potential down-stream signal molecule of the p210(BCR/ABL) tyrosine kinase in CML. The altered expression of cyclin D2 may contribute to the over proliferation of CML cells.

Blotting, Western ; Cyclin D2 ; Cyclins ; analysis ; genetics ; Flow Cytometry ; Genes, abl ; Humans ; K562 Cells ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction

OBJECTIVE: To investigate the expression and significance of regulatory T cells (Tregs), FoxP3 and transforming growth factor-β (TGF-β) in different phase of chronic myeloid leukemia (CML). METHODS: Peripheral blood of 73 CML patients in Department of Hematology, Heze Municipal Hospital from March 2018 to March 2021 were collected. According to patient's period in CML, they were divided into ND CML group (newly diagnosed), CP CML group (chronic period), and BP CML group (blast phase). The percentage of Tregs, expression level of FoxP3 mRNA and TGF-β were detected by flow cytometry, RT-qPCR, and ELISA, respecitively. The roles of above indices in clinical pathogenesis of patients with CML were analyzed. RESULTS: The proportion of Treg in the ND CML group was slightly higher than the CP CML group, but the difference was not statistically significant (P =0.695), while the BP CML group was significantly higher than the other two groups (P =0.008, P <0.001). The expression levels of FoxP3 mRNA in ND CML group, CP CML group and BP CML group were 11.61±2.21, 6.46±1.35 and 8.54±2.13, respectively. Significant difference in FoxP3 mRNA levels was observed among patients in different phases of CML (F =55.199, P <0.001). The expression levels of FoxP3 mRNA both in ND CML group and BP CML group were significantly higher than that in CP CML group (P <0.001), and the ND CML group was the highest (P <0.001). However, the expression levels of TGF-β in different phases of CML showed no statistical differences (H =0.634, P =0.728). CONCLUSION The abnormal distribution of Treg subset in different phases of CML and the significant increase of the expression level of FoxP3 mRNA in the new onset and blast phase of CML suggest that Tregs may promote the occurrence and progression of CML through immune regulation.
Humans ; Blast Crisis/metabolism* ; Forkhead Transcription Factors/metabolism* ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive/genetics* ; RNA, Messenger/metabolism* ; T-Lymphocytes, Regulatory/metabolism* ; Transforming Growth Factor beta/metabolism*

Chronic myeloid leukemia (CML) is a clonal myeloproliferative disorder characterized by a chromosome translocation that generates the Bcr-Abl oncogene encoding a constitutive kinase activity. Despite remarkable success in controlling CML at chronic phase by Bcr-Abl tyrosine kinase inhibitors (TKIs), a significant proportion of CML patients treated with TKIs develop drug resistance due to the inability of TKIs to kill leukemia stem cells (LSCs) that are responsible for initiation, drug resistance, and relapse of CML. Therefore, there is an urgent need for more potent and safer therapies against leukemia stem cells for curing CML. A number of LSC-associated targets and corresponding signaling pathways, including CaMKII-γ, a critical molecular switch for co-activating multiple LSC-associated signaling pathways, have been identified over the past decades and various small inhibitors targeting LSC are also under development. Increasing evidence shows that leukemia stem cells are the root of CML and targeting LSC may offer a curable treatment option for CML patients. This review summarizes the molecular biology of LSC and its-associated targets, and the potential clinical application in chronic myeloid leukemia.
Animals ; Chemokines ; metabolism ; Epigenesis, Genetic ; Humans ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; genetics ; metabolism ; pathology ; Neoplastic Stem Cells ; metabolism ; pathology ; Transcription Factors ; metabolism ; Tumor Microenvironment