OBJECTIVE: To explore the combined pro-apoptosis effect of HSP90 inhibitor BIIB021 and chloroquine (CQ) in chronic myeloid leukemia (CML) cells bearing T315I mutation and its mechanism. METHODS: The p210-T315I cells were divided into 4 groups by different treatment: control, BIIB021, CQ, and BIIB021 + CQ. After treated with BIIB021 or/and CQ for 24 hours, Annexin V/PI binding assay was used to detect apoptosis rates of CML cells. DAPI staining was used to observe nuclear fragmentation, and Western blot was used to detect the expression of caspase 3, PARP (apoptosis related proteins) and p62, LC3-I/II (autophagy related proteins). P210-T315I cells were inoculated subcutaneously into mice and CML mouse models were established. The mice in treatment groups were injected with BIIB021 and/or CQ while mice in control group were treated with PBS and normal saline. The tumor volume of mice was measured every 4 days, and protein level of cleaved-caspase 3 and LC3-II in tumor tissue were detected by immunohistochemistry. RESULTS: The results showed that BIIB021 induced apoptosis of CML cells in a dose-dependent manner ( r=0.91). CQ could enhance the apoptosis-inducing effect of BIIB021. Flow cytometry analysis results showed that the apoptosis rate of p210-T315I cells in combination group was higher than that in BIIB021 or CQ only group (P<0.05). DAPI staining showed nuclear fragmentation in combination group could be observed more obviously. Western blot analysis showed that BIIB021 could induce LC3-I to convert to LC3-II and decrease p62 protein levels (P<0.05). Moreover, the combination group had higher expression of LC3-II, p62 (P<0.05), activated PARP and activated caspase 3 than BIIB021 only group (P<0.05). Besides, experiment in vivo showed the mean tumor volume in co-treatment group was lower than that in single drug group (P<0.01). Immunohistochemistry of tumor tissue also showed the protein level of cleaved-caspase 3 and LC3-II in combined group was higher than that in BIIB021 only group. CONCLUSION HSP90 inhibitor BIIB021 induced significant apoptosis of CML cells bearing T315I both in vivo and in vitro. CQ can enhance this effect probably by autophagy inhibition.
Adenine/analogs & derivatives* ; Animals ; Apoptosis ; Autophagy ; Caspase 3/metabolism* ; Cell Line, Tumor ; Chloroquine/therapeutic use* ; Fusion Proteins, bcr-abl/pharmacology* ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive/drug therapy* ; Mice ; Mutation ; Poly(ADP-ribose) Polymerase Inhibitors/therapeutic use* ; Pyridines

OBJECTIVETo investigate the effect of steadily down-regulating the expression of VE-cadherin on the chemotheraputic sensitivity of K562 cells, and explore its possible mechanism.

METHODSSpecifically targeting interference sequences carrying human VE-cadherin were designed, the recombinant lentiviral vector containing the IRES-GFP and NEO segment was constructed; recombinant lentivirus was generated by three-plasmids packing system, and transfected into K562 cells, then the cells steadily down-regulated were sorted. CCK-8 assay was performed to evaluate the VE-cadherin of chemotherapeutic (Imatinib) sensitivity of K562 cells. The apoptosis was analyzed by flow cytometry with Annexin V/7-AAD double labeling. The expressions of CD133 and ALDH1 mRNA were determined by real time PCR. The protein expressions of VE-cadherin, BCR-ABL and β-catenin were analyzed by Western blot.

RESULTSThe recombinant lentiviral vector pLB-shVEC-NEO-IRES-GFP was successfully constructed, packed into the lentivirus, then the K562 cells steadily down-regulating VE-cadherin expression was obtained. When VE-cadherin was down-rengulated in K562 cells, the proliferation rate was reduced while the the apoptosis rate was increased; the mRNA levels of CD133 and ALDH1 also were reduced; BCR-ABL fusion protein was not obviously changed; the total β-catenin protein, as well as the nuclear β-catenin protein were decreased in the K562/shVEC cells. Conclution: K562 cells are more susceptible to chemotherapy when VE-cadherin is down-regulated, that may be realized via reducing the stability and the nuclear transfer of β-catenin protein.

Antigens, CD ; metabolism ; Apoptosis ; Cadherins ; metabolism ; Cell Proliferation ; Fusion Proteins, bcr-abl ; Humans ; K562 Cells

No abstract available.
Aged ; Cytidine Deaminase/*genetics/metabolism ; Dasatinib/therapeutic use ; Disease Progression ; Drug Resistance, Neoplasm ; Fusion Proteins, bcr-abl/genetics/metabolism ; Humans ; Imatinib Mesylate/*therapeutic use ; In Situ Hybridization, Fluorescence ; Karyotype ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive/*drug therapy ; Male ; Microfilament Proteins/*genetics/metabolism ; Oncogene Proteins/*genetics/metabolism ; Protein Kinase Inhibitors/*therapeutic use

Adenocarcinoma ; complications ; drug therapy ; metabolism ; Aged ; Antineoplastic Agents ; therapeutic use ; Fusion Proteins, bcr-abl ; metabolism ; Humans ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; diagnosis ; etiology ; metabolism ; Male ; Organoplatinum Compounds ; therapeutic use ; Stomach Neoplasms ; drug therapy ; metabolism

INTRODUCTIONThe prognosis of patients with chronic myeloid leukaemia (CML) has improved since the introduction of imatinib. However, patients who do not achieve complete cytogenetic response (CCyR) and major molecular response (MMR) have poorer prognosis. Recent clinical trials have demonstrated that early and deeper cytogenetic and molecular responses predict a better long-term outcome. This study aimed to analyse the relationship between early molecular response and clinical outcome in a real-life setting.

METHODSThis retrospective study included all patients with CML, in chronic or accelerated phase, who were treated with imatinib at University of Malaya Medical Centre, Malaysia.

RESULTSA total of 70 patients were analysed. The median follow-up duration was 74 months, and the cumulative percentages of patients with CCyR and MMR were 80.0% and 65.7%, respectively. Overall survival (OS) and event-free survival (EFS) at ten years were 94.3% and 92.9%, respectively. Patients who achieved CCyR and MMR had significantly better OS and EFS than those who did not. At six months, patients who had a BCR-ABL level ≤ 10% had significantly better OS and EFS than those who had a BCR-ABL level > 10%. The target milestone of CCyR at 12 months and MMR at 18 months showed no survival advantage in our patients.

CONCLUSIONOur data showed that imatinib is still useful as first-line therapy. However, vigilant monitoring of patients who have a BCR-ABL level > 10% at six months of treatment should be implemented so that prompt action can be taken to provide the best outcome for these patients.

Academic Medical Centers ; Adult ; Antineoplastic Agents ; therapeutic use ; Cytogenetics ; Disease-Free Survival ; Female ; Follow-Up Studies ; Fusion Proteins, bcr-abl ; metabolism ; Humans ; Imatinib Mesylate ; therapeutic use ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; diagnosis ; drug therapy ; genetics ; Malaysia ; Male ; Middle Aged ; Predictive Value of Tests ; Prognosis ; Retrospective Studies ; Treatment Outcome ; Universities

No abstract available.
Bone Marrow/pathology ; Chromosomes, Human, Pair 12 ; Chromosomes, Human, Pair 9 ; Core Binding Factor Alpha 2 Subunit/*genetics ; DNA/metabolism ; Gene Rearrangement ; Humans ; In Situ Hybridization, Fluorescence ; Karyotyping ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive/diagnosis/*genetics ; Male ; Middle Aged ; Oncogene Proteins, Fusion/*genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Translocation, Genetic

No abstract available.
Aged, 80 and over ; Base Sequence ; Bone Marrow/pathology ; DNA/chemistry/metabolism ; Female ; Fusion Proteins, bcr-abl/*genetics ; Humans ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive/diagnosis/*genetics ; Multiplex Polymerase Chain Reaction ; Protein Isoforms/genetics ; Sequence Analysis, DNA

OBJECTIVETo 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.

METHODSMTT 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.

RESULTSThe 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.

CONCLUSIONThe 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.

Apoptosis ; drug effects ; Cell Proliferation ; Down-Regulation ; Drug Resistance, Neoplasm ; Emodin ; analogs & derivatives ; pharmacology ; Fusion Proteins, bcr-abl ; metabolism ; Humans ; Imatinib Mesylate ; pharmacology ; K562 Cells ; drug effects ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; pathology ; Phosphorylation

OBJECTIVETo study the signal patterns of dual color dual fusion fluorescence in situ hybridization (DCDF-FISH) for detection of genetic abnormality in adult acute lymphoblastic leukemia (ALL) patients and their diagnostic value and clinical application.

METHODSThe clinical data of 68 ALL patients confirmed in our hospital were analyzed retrospectively; The bone marrow samples were detected by DCDF-FISH, flow cytometry, conventional cytogenetics (CCG), reverse transcriptase polymerase chain reaction (RT-PCR), and the correlation of these results was compared. And the reaction of patients to treatment was dynamically observed by DCDF-FISH.

RESULTSSixteen signal patterns were found in DCDF-FISH, including 14 kinds of atypical signal patterns (signal patterns of 1R2G, 2R3G, 2R4G and 3R3G as abnormal signal patterns without BCR/ABL fusion gene. Signal patterns of 1R1G1F, 1R1G3F, 1R1G4F, 1R2G1F, 1R2G2F, 1R2G3F, 1RnG2F (n ≥ 3), 2R2G1F, 1G4F, 1R4F corresponded to t (9;22) karyotype). Ph(+) ALL patients accounted for 17. All cases with Ph chromosome or BCR/ABL positive were B-ALL or My(+)-B-ALL. The Ph chromosome was detected in 12 cases (positive rate was 18%) by CCG. The positive rate was 25% (17/68) by DCDF-FISH and RT-PCR. The DCDF-FISH fluorescence pattern change before and after chemotherapy of the patients showed that the quantity and form of the signal pattern was changed after chemotherapy, and the common characteristics was the Ph chromosome in patients.

CONCLUSIONThe DCDF-FISH is a sensitive and reliable method for the detection of BCR/ABL rearrangement. Analyzing the dynamical change of DCDF-FISH signal patterns has been comfirmed to have a important guiding significance in the diagnosis, and anlysis of response to therapy, drug resistance and the prognosis of ALL patients.

Bone Marrow ; metabolism ; Flow Cytometry ; Fusion Proteins, bcr-abl ; genetics ; Gene Rearrangement ; Humans ; In Situ Hybridization, Fluorescence ; Karyotyping ; Philadelphia Chromosome ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; diagnosis ; genetics ; Reverse Transcriptase Polymerase Chain Reaction

OBJECTIVETo explore the differences of clinical characteristics and outcome between p190 and p210 transcripts in adult Ph chromosome positive acute lymphoblastic leukemia patients in the new era with tyrosine kinase inhibitor (TKI) treatment, so as to provide an insight for improving the prognostic stratification and individualized treatment of the Ph(+) ALL patients.

METHODSThe clinical data of 65 patients were analysed retrospectively, these patients were diagnosed as Ph(+) ALL and treated with conventional chemotherapy plus TKI treatment with or without hematopoietic stem cell transplantation (HSCT) from January 2005 to December 2014 in our hospital, then the differences of clinical features and prognosis were compared between the p190 (n = 41) and the p210 group (n = 24).

RESULTSThe p190 group had lower platelet count than the p210 group (46.3 × 10(9)/L vs 65 × 10(9)/L) (P = 0.084); the leukemic blast cells in bone marrow at diagnosis was slightly higher in p190 group than that in p210 group (88.4% vs 76.8%) (P = 0.096); the other clinical features, such as sex, age, white blood cells, hemoglobin, leukemic blast cells in peripheral blood, and BCR-ABL/ABL expression level were not significantly different between these two groups. As to the response to treatment, the complete remission rate (CR) after induction therapy was 80% (32/40) and 87% (20/23) respectively in the p190 and p210 group, no significant difference was seen (P = 0.732). The time from induction to the first complete remission (CR1) was not significantly different either (28 days vs 29 days) (P = 0.922). The recurrence rate was 61% (20/33) in the p190 group and 43% (9/21) in the p210 group, but the difference was not significantly different (P = 0.202). However, the duration of remission in p190 group was shorter than that in p210 group, whether from the time of initial diagnosis to relapse (212 days vs 274 days) (P = 0.077) or from the time of CR1 to relapse (146 days vs 242 days) (P = 0.084). For the prognosis, the p190 group presented with a shorter 5 year-survival rate (P = 0.016) as well as event-free survival rate (P = 0.085).

CONCLUSIONThe p190 group tends to have lower peripheral blood platelet count and higher percentage of leukemic blasts in bone marrow at diagnosis; while the CR rate and the time from induction to CR1 are not significantly different; however, the p190 group is more likely to relapse at a relatively early stage, and the 5 year-survival rate and event-free survival rate are lower in p190 group than that in p210 group, indicating that the patients carrying p190 transcript are probably necessary to receive more intensive therapy such as HSCT as early as possible after achieving CR1, which can promisingly improve the overall prognosis of the Ph(+) ALL patients.

Acute Disease ; Disease-Free Survival ; Fusion Proteins, bcr-abl ; genetics ; metabolism ; Hematopoietic Stem Cell Transplantation ; Humans ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; drug therapy ; genetics ; metabolism ; Prognosis ; Protein Kinase Inhibitors ; therapeutic use ; Recurrence ; Remission Induction ; Retrospective Studies ; Survival Rate