[Role of WAVE1 in K562 leukemia cells invasion and its mechanism].

OBJECTIVE: To investigate role of WASP family verprolin homologous protein 1 (WAVE1) in K562 leukemia cell invasion and its mechanism. METHODS: Immunofluorescence method was used to detect the distribution of WAVE1 and MMP-2 in the cells. K562 cells were transfected with pcDNA3. 1-WAVE1 reconstructed plasmid or with specific siRNA to WAVE1 gene. The invasion ability of K562 cells was examined by Transwell assay. The expression level of WAVE1 and MMP-2 in K562 cells was assayed by real-time PCR and Western blot. RESULTS: (1) WAVE1 and MMP-2 mainly expressed and co-localized in the cell membrane; (2) 24 h and 48 h after transfected with pcDNA3. 1-WAVE1, the MMP-2 mRNA level in K562 cells increased by 295% and 198% while its protein increased by 80% and 23% respectively as compared with control K562 cells. At the same time point after transfected with specific siRNA, the MMP-2 mRNA level decreased by 81% and 28%, and its protein decreased by 36% and 53% respectively as compared with control. (3) The invasion ability of K562 cells was enhanced after transfected with pcDNA3. 1-WAVE1 and depressed after transfected with the specific siRNA. CONCLUSION: The co-localization of WAVE1 and MMP-2 in K562 cells suggests they coordinate in functions; WAVE1 may involve in the migration and invasion of K562 cells through regulating the expression level of MMP-2.

[Expression of WAVE1 and p22phox in children with acute lymphocytic leukemia and the relationship of WAVE1 with oxidative stress].

OBJECTIVE: To study the expression of WAVE1 and p22phox in peripheral blood mononuclear cells (PBMCs) in children with acute lymphocytic leukemia (ALL) and the relationship of WAVE1 with oxidative stress. METHODS: Real-time PCR was used for detecting WAVE1 and p22phox expression in PBMCs in 41 children with ALL and 10 normal controls. Plasma activity of superoxide dismutase (SOD) was measured by the xanthine oxidase method. Plasma activity of GSH-Px was measured by the DTNB reaction test. RESULTS: The expression of WAVE1 and p22phox was significantly higher in the active ALL groups (newly diagnosed and relapse ALL) than that in the normal control and the complete remission (CR) ALL groups (<0.01). The CR ALL group showed increased WAVE1 and p22phox expression than those in the normal control group (<0.05). Plasma activities of SOD (22.62+/-7.39 U/mL) and GSH-Px (91.73+/-28.88 micromol/L) in the active ALL group were significantly lower than those in the normal control (166.35+/-27.93 U/mL and 490.94+/-39.38 micromol/L, respectively) and the CR ALL groups (107.11+/-28.57 U/mL and 267.56+/-82.64 micromol/L, respectively) (<0.01). WAVE1 expression was positively correlated with p22phox expression (r=0.34, <0.05) but negatively correlated with plasma activities of SOD and GSH-Px ( r=-0.336 and-0.408, respectively; <0.05). CONCLUSIONS: WAVE1 and p22phox expression in PBMCs increased and was associated with the disease course in children with ALL. Oxidative stress may be involved in the regulation of WAVE1 expression in ALL children.

[Efficacy and prognosis analysis in 188 children with acute lymphoblastic leukemia of China].

OBJECTIVE: To analyze the therapeutic effect and the influencing factors of event-free survival (EFS) of childhood acute lymphoblastic leukemia (ALL) in Xiangya Hospital of Central South University and the First Affiliated Hospital of Guangxi Medical University. METHODS: All the patients adopted chemotherapy according to therapeutic guideline revised by the Subspecialty Group of Hematology, The Society of Pediatrics, Chinese Medical Association for the second-time in 1998 (the Rongcheng ALL-98 Protocol). Kaplan-Meier method was used to estimate the survival rates of 188 patients who received therapy with good compliance. The differences of EFS between groups were assessed by Log-rank test. The independent influencing factors on EFS were analyzed by the Cox proportional hazards regression model. RESULTS: After receiving inductive treatment, 354 of 374 (93.6%) patients demonstrated a complete remission; 188 patients who received complete courses of treatment with good compliance showed (68.1 +/- 5.6)% five-year EFS. Meanwhile, the five-year EFS in standard-risk (SR) group and high-risk (HR) group were (75.2 +/- 6.0)% and (47.6 +/- 11.6)%, respectively. The total relapse rate was 10.6% and the median time to relapse was 13 months. Twenty-nine of 188 patients (15.4%) were dead, and 13 patients (7.0%) died from treatment-related complications. Independent adverse prognostic factors included risk grouping, t (9; 22)/bcr-abl gene and leukocyte count. CONCLUSIONS: The total EFS of childhood ALL patients treated with Rongcheng ALL-98 Protocol in two hospitals was close to 70%. Therefore, it is necessary to evaluate risk factors and consider the grouping in more detail to reduce the treatment-related mortality and to increase the compliance of treatment which can ultimately improve the EFS.

[Enhancive effect of HMGB1 gene silence on adriamycin-induced apoptosis in K562/A02 drug resistance leukemia cells].

OBJECTIVE: To investigate the effect of high mobility group boxl (HMGBI) gene silence on adriamycin (ADM)-induced apoptosis in K562/A02 drug resistance leukemia cells. METHODS: K562/ A02 cells were transient transfected with HMGB1- small interference RNA(siRNA) vector, and the levels of HMGB1 gene differential expression pre-and post-transfection were measured by RT-PCR and Western blotting. 50% inhibition concentration (IC50) of ADM on K562/A02 was determined by WST-8 assay. Cell apoptosis was assessed by flow cytometry. The release of Smac/DIABLO from the mitochondria to the cytoplasm was assayed by Western blotting. Activity of Caspase-3 was assayed with a Caspase Colorimetric Assay Kit. RESULTS: (1) The HMGB1 expression at mRNA and protein levels in HMGB1 siRNA transfected K562/A02 cells were decreased by 86% and 71% respectively compared with control. (2) Suppression of HMGB1 by siRNA in K562/A02 cells resulted in a reversal of the resistance to ADM, and decreased IC50 from (4.83 +/- 0.08) microg/ml to (1.33 +/- 0.10) microg/ml. 1 microg/ml and 5 microg/ml of ADM treatment increased cell apoptotic rate by 27% and 32% respectively. (3) HMGB1 suppression in K562/A02 cells significantly promoted ADM- induced Smac/DIABLO release from the mitochondria to the cytoplasm, and increased the activities of Caspase-3. CONCLUSION: HMGB1 gene silence can enhance sensitivity of K562/A02 cells to ADM and reverse cell resistant to ADM.

[Expression of WAVE1 in childhood acute lymphocytic leukemia and in the apoptosis of Jurkat cells induced by adriamycin].

OBJECTIVE: To investigate whether WASP/Verprolin homologous protein 1 (WAVE1) plays a role in the pathogenesis of childhood acute lymphoblastic leukemia (ALL). METHODS: WAVE1 mRNA and protein expression in bone marrow mononuclear cells (BMMCs) was measured by RT-PCR and Western blotting respectively in 4 children with ALL relapse, 15 children with ALL in complete remission (CR) and 40 children with newly diagnosed ALL. Ten normal bone marrow samples were used as controls. Jurkat cells were treated with different concentrations of adriamycin (ADM). The cell proliferation was detected with MTT. The apoptosis rate was measured by flow cytometry. WAVE1 mRNA and protein expression of Jurkat cells treated with ADM was detected by RT-PCR and Western blotting respectively. RESULTS: WAVE1 was not expressed or weakly expressed in BMMCs from normal controls and patients with ALL in CR. Higher WAVE1 mRNA and protein expression was found in BMMCs from patients with newly diagnosed ALL and patients with relapse ALL when compared with the controls and the patients in CR (P<0.01). ADM significantly inhibited the proliferation of the Jurkat cells and the inhibitory effect was dose-and time-dependent (P<0.05). After ADM treatment for 24 hrs, the percentage of apoptosis cells increased significantly and WAVE1 mRNA and protein expression of Jurkat cells decreased significantly when compared with the untreated controls (P<0.05). CONCLUSIONS: The WAVE1 expression increased in children with ALL. WAVE1 may be related to the development of ALL and may be severed as a marker for the evaluation of the severity of ALL in children.

[Role of high mobility group box 1 in adriamycin-induced apoptosis in leukemia K562 cells].

BACKGROUND & OBJECTIVE: High mobility group box l (HMGB1), a nuclear DNA-binding protein, stabilizes the structure and function of chromatin, regulates gene transcription. Recent studies found that HMGB1 is associated with the proliferation and metastasis of many tumors, including breast cancer, colon carcinoma, and melanoma, and is rich in various solid cancer tissues and immature cells. This study was to explore the role of HMGB1 in adriamycin (ADM)-induced apoptosis in leukemia K562 cells. METHODS: K562 cells were transiently transfected with recombinant plasmid pcDNA3.1-HMGB1. The expression of HMGB1 in K562 cells were detected by Western blot and reverse transcription-polymerase chain reaction (RT-PCR). The 50% inhibition concentration (IC(50)) of ADM for K562 cells was determined by WST-8 assay. Cell apoptosis was assessed by flow cytometry. The protein level of Bcl-2 was detected by Western blot. The activities of Caspase-3 and Caspase-9 were assayed with Caspase Colorimetric Assay Kit. RESULTS: The mRNA and protein levels of HMGB1 in K562 cells transfected with pcDNA3.1-HMGB1 were increased by about 85% and 56% respectively as compared with those in K562 cells transfected with pcDNA3.1. Overexpression of HMGB1 in K562 cells by transient transfection significantly increased the resistance to ADM; the IC(50) of ADM was increased from (0.06+/-0.00) microg/mL to (3.46+/-0.06) microg/mL. When treated with 1 microg/mL ADM, the apoptosis rate was significantly lower in HMGB1-transfected K562 cells than in pcDNA3.1-transfected K562 cells [(12.00+/-1.26)% vs. (44.50+/-1.87)%, P<0.05]. Overexpression of HMGB1 in K562 cells significantly inhibited ADM-induced down-regulation of Bcl-2 protein. After treatment of ADM, the activities of Caspase-3 and Caspase-9 in HMGB1-transfected K562 cells were inhibited as compared with those in pcDNA3.1-transfected K562 cells (Caspase-3: 1.55+/-0.06 vs. 2.55+/-0.06 at 12 h, 1.86+/-0.10 vs. 2.85+/-0.06 at 24 h, P<0.05; Caspase-9: 1.40+/-0.08 vs. 2.03+/-0.05 at 12 h, 1.55+/-0.06 vs. 2.22+/-0.05 at 24 h, P<0.05). CONCLUSION: HMGB1 overexpression could inhibit ADM-induced apoptosis in leukemia K562 cells through regulating the protein level of Bcl-2 and the activities of Caspase-3 and Caspase-9.

[Role of WAVE1 in drug resistance of K562/A02 leukemia cells].

OBJECTIVE: To investigate if WAVE1 is involved in mult drug-resistance (MDR) of human leukemia cell line K562/A02. METHODS: The level of WAVE1 in K562 and K562/A02 cells was assayed by Western blot and RT-PCR; K562 cells and K562/A02 cells were transient transfected with pEFBOS-WAVE1 reconstructed plasmid or specifically siRNA to WAVE1. 50% inhibition concentration (IC50) of doxorubicin on K562/A02 was determined by WST-8 assay. Hoechst33258 staining was used to examine cell morphological changes and to calculate percentage of apoptotic nuclei. The mRNA level of mdrl was assayed by RT-PCR. The Bcl-2 protein was assayed by Western blot. RESULTS: 1. The WAVE1 expression at mRNA and protein level in K562/A02 cells was increased by about 70% and 63% respectively as compared with that in K562 cells. 2. Overexpression of WAVE1 in K562 cells by transient transfection significantly increased the resistance to doxorubicin, and increased IC50 from (0.05 +/- 0.00) microg/ml to (2.99 +/- 0.12) microg/ml, and at 1 microg/ml or 5 microg/ml of doxorubicin treatment, cell apoptotic nuclei rate was decreased by 30% or 35% respectively. 3. Suppression of WAVE1 in K562/A02 cells by siRNA resulted in a reversal of MDR to doxorubicin, and decreased IC50 from (4.29 +/- 0.15) microg/ml to (1.85 +/- 0.07) microg/ml, and at 1 microg/ml or 5 microg/ml of doxorubicin treatment, cell apoptotic nuclei rate was increased by 24% or 21% respectively. 4. Overexpression of WAVE1 in K562 cells significantly increased the mdrl mRNA and the Bcl-2 protein, and suppression of WAVE1 in K562/A02 cells by siRNA decreased the mRNA and the protein. CONCLUSION: WAVE1 involves in the MDR mechanisms in K562/A02 leukemia cells through regulation the level of mdrl and Bcl-2.