OBJECTIVETo study the gene expression profile of chronic myeloid leukemia (CML) in chronic and blast phases for providing information about the potential mechanisms of blastic crisis.

METHODSThe gene expression difference of bone marrow mononuclear cells between CML in chronic and in blastic phases was examined with DNA array.

RESULTSIn the blastic phase, 68 genes were obviously up-regulated in the 1176 tested genes. Among the 68 genes, the transcription factors accounted for 23.5%, cell surface antigens 22.1%, regulation proteins 19.1% and others 35.3%. There were 17 genes expressed only in the blastic phase and 11 (16.2%) genes related to G protein.

CONCLUSIONSObvious difference of gene expression profile existed between chronic and blastic phases of CML. Up-regulation of gene expression is one of the characteristics of CML in blastic phase. The genes related to G protein may play an important role in the blastic transformation of CML.

Blast Crisis ; genetics ; metabolism ; Bone Marrow Cells ; metabolism ; Cells, Cultured ; Female ; Gene Expression Profiling ; Gene Expression Regulation, Neoplastic ; Humans ; Male

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*

The purpose of this study was to investigate the growth characteristics and the expression level of integrin mRNA of the cultured bone marrow mesenchymal stem cells (BMMSCs) from patients with chronic myeloid leukemia (CML) in myeloid crisis (MC), and explore the role of BMMSCs in pathogenesis of CML. Five CML patients were enrolled in experimental group, five healthy persons were used as control. BMMSCs were cultured in vitro. The morphology of BMMSCs was observed every day and the growth curve were portrayed, and the ability of cell proliferation were detected according to the daily results of cell counting. Total RNA was extracted from third and fourth passages of BMMSCs, The expression of integrins mRNA of BMMSCs were measured by real-time PCR. The results showed that the BMMSCs of experimental and control groups had no difference in growth characterisctics, but the expression of integrins mRNA of the BMMSCs was higher in CML patients than in normal control group (p < 0.05). It is concluded that the abnormally high expression of integrins of BMMSC from the CML patients take part in pathogenesis of CML.
Adult ; Blast Crisis ; metabolism ; Bone Marrow Cells ; metabolism ; pathology ; Cell Proliferation ; Female ; Humans ; Integrins ; genetics ; metabolism ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; metabolism ; pathology ; Male ; Mesenchymal Stromal Cells ; metabolism ; pathology ; Middle Aged ; RNA, Messenger ; genetics ; metabolism ; Tumor Cells, Cultured

The aim of this study was to investigate the expression status of the helicase antigen (HAGE) transcript and its clinical significance in patients with acute myeloid leukemia (AML) and chronic myeloid leukemia (CML). The expression of HAGE cDNA in bone marrow mononuclear cells from AML and CML patients was detected by using real-time quantitative PCR. The results indicated that overexpression of HAGE transcript (117.12% - 9842.70%, median 434.96%) was detected in 14.8% (11/74) AML patients. AML patients with HAGE cDNA expression were significantly older than those HAGE-negative patients (median 67 and 45 years, respectively, p = 0.001). HAGE cDNA expression was more frequently present among the patients with acute monoblastic leukemia (M(4) and M(5), 7 of 20, 35.0%), compared to the patients with acute non-monoblastic leukemia (M(1), M(2), M(3) and M(6), 4 of 54, 7.4%) (p = 0.007). 28.6% (8/28) cases with normal karyotypes showed HAGE cDNA overexpression, significantly higher than 7.5% (3 of 40) in those with chromosomal abnormalities (p = 0.041). Overexpression of HAGE transcript was found in 9 (34.6%) CML cases and more frequently observed at accelerated phase and blast crisis (4/4, 100%) than that at chronic phase (5/22, 22.7%) (p = 0.008). It is concluded that HAGE cDNA expression is relevant to specific subtypes of AML and to the progression of CML.
Blast Crisis ; Bone Marrow Cells ; metabolism ; DEAD-box RNA Helicases ; genetics ; metabolism ; DNA, Complementary ; Gene Expression ; Humans ; Karyotype ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; genetics ; metabolism ; Leukemia, Myeloid, Acute ; genetics ; metabolism ; Neoplasm Proteins ; genetics ; metabolism ; RNA, Messenger ; genetics

OBJECTIVETo investigate the expression and the possible mechanism of the transcription factor C/EBPα in chronic myeloid leukemia（CML).

METHODSBone marrow samples from 50 CML patients（including 33 patients in chronic phase, 7 in accelerated phase and 10 in blast crisis）and peripheral blood specimens of 20 healthy donors were collected. The expression of C/EBPα gene and the effect of Imatinib on its expression was detected by RT- PCR. C/EBPα gene was inserted into lentivirus expression vector pLVX- EGFP- 3FLAG- Puro by recombinant DNA technology to construct C/EBPα stable expression in K562 cells. Cell proliferation was assayed by CCK-8. The expressions of Foxo3a and Bim genes were detected by RT-PCR.

RESULTSThe level of C/EBPα expression was significantly declined in CML patients compared with that of normal control group（P<0.01）and had negative correlation with bcr- abl expression（Spearman r=－ 0.505, P<0.01). The stable K562- C/EBPα cell line was successfully established and confirmed by RT-PCR and Western blot. Cell proliferation ability was lower in the K562- C/EBPα group than that in the non- transfection and mock-vehicle groups. The expressions of Foxo3a and Bim genes were 1.06 ± 0.06 and 0.53 ± 0.07, respectively, which was higher than that of nontransfection and mock-vehicle groups（P<0.01, P<0.05).

CONCLUSIONC/EBPα expression was decreased in CML patients, overexpression of C/EBPα could inhibit K562 cell growth.

Blast Crisis ; Bone Marrow ; CCAAT-Enhancer-Binding Protein-alpha ; metabolism ; Case-Control Studies ; Cell Cycle ; Cell Proliferation ; Humans ; Imatinib Mesylate ; K562 Cells ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; metabolism ; Transfection

OBJECTIVETo evaluate the efficiency of dasatinib as the second- or third-line tyrosine kinase inhibitor (TKI)in imatinib-resistant patients with chronic myeloid leukemia (CML)based on BCR-ABL mutation detection.

METHODS122 CML patients received dasatinib treatment, including 83 with imatinib-resistance and 39 with both imatinib- and nilotinib-resistance, 55 in the chronic-phase (CP), 21 in the accelerated- phase (AP)and 46 in the blast- phase (BP). Those harboring dasatinib highly- resistant mutations (T315I/A, F317L/V/C and V299L)were excluded based on BCR-ABL kinase domain mutation screening by Sanger sequencing at baseline. Hematologic, cytogenetic and molecular responses were evaluated regularly, and rates of progression-free-survival (PFS)and overall survival (OS)were analyzed. BCR- ABL mutation detection was performed once the patients failed on dasatinib.

RESULTSIn the CP patients, the rates of complete hematological response (CHR), complete cytogenetic response (CCyR), major molecular response (MMR)and molecular response 4.5 (MR4.5)were 92.7%, 53.7%, 29.6% and 14.8%, respectively. 4-year PFS and OS rates were 84.4% and 89.5%, respectively. In the AP patients, HR and CCyR rates were 81.0% and 35.0%; and 3-year PFS and OS rates were 56.1% and 59.3%, respectively. In the BP patients, HR and CCyR rates were 63.0% and 21.4%; and 1-year PFS and OS rates were 43.6% and 61.8%, respectively. Outcomes were similar when dasatinib was used as the second- line TKI or the third-line TKI. Of the 75 patients who were resistant to dasatinib, 37 (48.7%)developed new mutation(s), and T315I (59.5%)was the most common mutation type. The patients who already harbored mutation(s)before dasatinib therapy achieved similar responses and outcomes to those with no mutation at baseline. However, they had higher likelihood of developing additional mutations associated with resistance to dasatinib (65.7%vs 34.1%,P=0.006).

CONCLUSIONSDasatinib was proved to be effective in the treatment of imatinib- or/and nilotinib-resistant CML patients, especially in both CP and AP cohorts. The significance of BCR-ABL mutation screening and monitoring should be highlighted before and during dasatinib therapy.

Blast Crisis ; Cytogenetics ; Dasatinib ; therapeutic use ; Disease-Free Survival ; Drug Resistance, Neoplasm ; Fusion Proteins, bcr-abl ; metabolism ; Humans ; Imatinib Mesylate ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; drug therapy ; Mutation ; Protein Kinase Inhibitors ; therapeutic use ; Pyrimidines

OBJECTIVETo identify and compare the expression profiles of differential proteins between chronic phase and blast crisis in chronic myeloid leukemia (CML) by proteomic analysis, and screen the proteins related to blast crisis.

METHODSThe total cellular proteins from the bone marrow cells at chronic phase (CP) and blast crisis (BC) in CML were separated by two-dimensional polyacrylamide gel electrophoresis (2-DE) and analyzed with ImageMaster 5.0 software to screen the differential protein spots. Differential protein spots were identified by mass spectrometry for peptide mass fingerprint in combination with database searching from SWISS-PROT. Then 3 protein spots were selected to verify at protein and mRNA levels by Western blot and semi-quantitative RT-PCR, separately.

RESULTSComparing gel pages from CML-CP and CML-BC, the expression of 13 protein spots decreased and 25 protein spots increased significantly in CML-BC. Twenty differential protein spots were identified by mass spectrometry and 15 were successfully determined. The results of Western blotting were similar to those of 2-DE and showed a high expression of hnRNPK, annexin A1 and RhoA. Semi-quantitative RT-PCR analysis showed that there was no correlation between the protein expression changes and mRNA levels of hnRNPK, annexin A1 and RhoA.

CONCLUSIONA group of proteins associated with blast crisis are obtained and the results may provide clues for further research to elucidate the role of these proteins in CML-BC carcinogenesis and to develop potential associated biomarkers.

Adult ; Aged ; Annexin A1 ; genetics ; metabolism ; Blast Crisis ; genetics ; metabolism ; Female ; Gene Expression Profiling ; Heterogeneous-Nuclear Ribonucleoprotein K ; Humans ; Leukemia, Myeloid, Chronic-Phase ; genetics ; metabolism ; pathology ; Male ; Middle Aged ; Proteomics ; methods ; RNA, Messenger ; metabolism ; Ribonucleoproteins ; genetics ; metabolism ; Young Adult ; rhoA GTP-Binding Protein ; genetics ; metabolism

OBJECTIVETo investigate the underlying mechanism and clinical significance of PU.1 down-expression in chronic myeloid leukemia (CML) patients.

METHODSDifferent methylation status of PU.1 promoter region containing 20 CpG islands in normal individuals, CML chronic phase and blast crisis patients, complete cytogenetic remission patients after imatinib treatment, and blast crisis bone marrow K562 CML cells was detected by bisulfite sequencing. Semi-quantitative PCR was used to detect the PU.1 mRNA expression in normal controls, CML chronic phase and blast crisis patients, and blast crisis bone marrow K562 CML cells. Indirect immune fluorescence and Western blot were used to analyze the exprtession of PU.1 protein in normal individuals, CML chronic phase and blast crisis patients, and blast crisis bone marrow K562 CML cells.

RESULTSAberrant methylation in the promoter region of transcription factor PU.1 was found in both CML chronic phase and blast crisis phase bone marrow cells, as well as in CML blast K562 cells. Down-expression of PU.1 mRNA and protein levels was found in above cells. No methylation in the promoter region of PU.1 was observed in normal individuals, and the PU.1 mRNA and protein expressions were not reduced at all. Furthermore, high methylation status of bone marrow cells was even observed in the CML patients who acquired complete cytogenetic remission.

CONCLUSIONSThe results of our study indicate that the epigenetic modification of PU.1 in CML patients and K562 cell line might be responsible for the down-expression of PU.1. The data suggest that aberrant methylation of PU.1 plays a role in CML pathogenesis, therefore, it might serve as a useful biomarker and potential target in therapy for chronic myeloid leukemia.

Antineoplastic Agents ; therapeutic use ; Benzamides ; Blast Crisis ; Bone Marrow Cells ; metabolism ; pathology ; CpG Islands ; genetics ; DNA Methylation ; Down-Regulation ; Epigenesis, Genetic ; Gene Expression Regulation, Leukemic ; Humans ; Imatinib Mesylate ; K562 Cells ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; drug therapy ; genetics ; metabolism ; Piperazines ; therapeutic use ; Promoter Regions, Genetic ; genetics ; Proto-Oncogene Proteins ; genetics ; metabolism ; Pyrimidines ; therapeutic use ; RNA, Messenger ; metabolism ; Trans-Activators ; genetics ; metabolism

OBJECTIVETo investigate the changes in the expression of beta-catenin in patients with chronic myeloid leukemia (CML) in different phases, and explore the relationship between beta-catenin and the cytogenetic response to imatinib mesylate.

METHODSBeta-catenin mRNA and protein expressions were detected by RT-PCR and Western blotting in the bone marrow mononuclear cells (BMMNCs) from 99 CML patients. The expressions of BCR-ABL fusion gene at both the mRNA and protein levels were detected by fluorescence in situ hybridization (FISH) in 94 patients before and during the one-year treatment with imatinib mesylate at the interval of 3 months, and the relationship between beta-catenin and cytogenetic response to imatinib mesylate was analyzed.

RESULTSThe expression of beta-catenin increased significantly in patients with blast crisis and accelerated phase (P<0.001), but showed no significant difference between normal subjects and CML patients in the chronic phase (P>0.05). The main cytogenetic remission rate was significantly higher in patients who were consistently negative for beta-catenin than in those consistently positive for beta-catenin or those with a positive transformation (P<0.001).

CONCLUSIONBeta-catenin overexpression in the progression of CML, consistent high level of beta-catenin or a positive transformation may indicate a poor response to imatinib, and early measures should be taken to increase the remission rate.

Adolescent ; Adult ; Benzamides ; therapeutic use ; Blast Crisis ; drug therapy ; genetics ; metabolism ; Case-Control Studies ; Female ; Humans ; Imatinib Mesylate ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; drug therapy ; genetics ; metabolism ; pathology ; Male ; Middle Aged ; Piperazines ; therapeutic use ; Pyrimidines ; therapeutic use ; RNA, Messenger ; genetics ; Young Adult ; beta Catenin ; metabolism