OBJECTIVE: To establish a serum-free culture system of dendritic cells (DCs) from chronic myeloid leukemia (CML) cells so that DCs vaccine may be applied to the adoptive immunotherapy of CML in the near future. METHOD...OBJECTIVE: To establish a serum-free culture system of dendritic cells (DCs) from chronic myeloid leukemia (CML) cells so that DCs vaccine may be applied to the adoptive immunotherapy of CML in the near future. METHODS: Fetal calf serum, serum-free medium and autologous serum were used for culture of DCs. The usage of cytokines was classified into two groups: group A (stem cell factor, granulocyte/macrophage colony-stimulating-factor, tumor necrosis factor-alpha and interleukin-4) and group B (granulocyte/macrophage colony-stimulating-factor, tumor necrosis factor-alpha and interleukin-4). The phenotypes of DCs were analyzed by using indirect immunofluorescence and flow cytometry. Mixed leukocyte responses were performed by methyl thiazolyl tetrazolium (MTT) assay. Chromosome analysis of DCs can be achieved by displaying G banding. T cells from CML patients were stimulated with autologous DCs and T-cell cytotoxicity was measured by (MTT) assay. RESULTS: CD34(+) cells or mononuclear cells were obtained from peripheral blood or bone marrow samples of eight patients of chronic-phase CML. Group A of serum-free medium was better than group B in expansion of total cell numbers and the rate of DCs. These results of serum-free medium were not significantly different from those of fetal calf serum medium, but the results of autologous serum medium were inferior to two groups above. The expression of major histocompatibility complex class II antigen on the surface of DCs was notable (> 50%), but the expression of CD83 and the costimulatory molecules CD86 was not noticeable (10% - 50%). Although CD1a(+)/CD14(-) DCs were potent stimulators of allogeneic lymphocytes, expansion of T cells from normal volunteers were not significant (average 27.2 fold at DCs: T cells ratio of 1:10). At day 12, CD1a(+) cells from three patients were studied by displaying G banding and Ph(+) cells in these populations were 100%, 98% and 60%, respectively. At an effector: target ratio of 40:1, 32% to 45% cytotoxicity was noted with DC-stimulated T cells against autologous leukemia cells. CONCLUSIONS: A stable serum-free culture system of CML-DCs was established. The expression of CD83 and CD86 on the surface of CML-DCs and DCs' potent stimulation of allogeneic lymphocytes were not notable. DCs in CML patients can be derived from the malignant clone and these malignant DCs could induce anti-leukemic reactivity in autologous T lymphocytes without the necessity for additional exogenous antigens.展开更多
OBJECTIVE To investigate the effects of survivin antisense oligodeoxy-nucleotid (ASODN) on proliferation and apoptosis in the chronic myeloid leukemia cell line K562. METHODS Different concentrations of an antisense o...OBJECTIVE To investigate the effects of survivin antisense oligodeoxy-nucleotid (ASODN) on proliferation and apoptosis in the chronic myeloid leukemia cell line K562. METHODS Different concentrations of an antisense oligodeoxy-nucleotid and control sequence (scrambled ODN) targeting the survivin gene were transferred into K562 by a lipofectin reagent. The MTT assay was used to measure the growth inhibitory rate, IC50, and to observe the cytotoxicity of survivin ASODN in the K562 cells. The morphologic changes in the nucleus and the apoptotic rate were observed by Hoechst33342/PI staining. Caspase-3 activity was evaluated by a kinase activity assay. The changes of survivin protein expression after transfection were detected by Western blots. RESULTS Eight hours after transfection, fluorescence in the K562 cells was well distributed. Treatment of the cells for 44 h with different concentrations of survivin ASODN produced a IC50 of 800 nmol/L. The growth inhibitory rate with 200, 400, 600 and 1000 nmol/L of survivin ASODN was 15.8±1.6%, 23.8±5.9%, 37.1±5.6% and 77.3±2.5% respectively. After 36 h of of survivin ASODN treatment, distinct morphologic changes characteristic of cell apoptosis such as karyopyknosis and conglomeration were observed by Hoechst33342/PI staining. Caspase-3 activity increased significantly after treatment of the cells with different concentrations of survivin ASODN(P<0.01)and following treatment with 800 nmol/L survivin ASODN, survivin expression decreased significantly. CONCLUSION Survivin ASODN exerts an anti-cancer effect by inducing apoptosis in K562 leukaemia cells. Up-regulated expression of caspase-3 may play a role in this process.展开更多
Background Arsenic trioxide (As2O3) has been identified as a very potent antiacute leukemic agent However its role in apoptosis needs to be elucidated As2O3 interferes with the proliferation and survival of tumor cell...Background Arsenic trioxide (As2O3) has been identified as a very potent antiacute leukemic agent However its role in apoptosis needs to be elucidated As2O3 interferes with the proliferation and survival of tumor cells via a variety of mechanisms Drugtarget interactions at the level of nuclear matrix (NM) may be critical events in the induction of cell death by As2O3 This study dealt with As2O3-target interactions at the level of NM in chronic myelogenous leukemia cell line K562 by proteomics Methods K562 cells were cultured in MEM and treated with different concentrations of As2O3 The nuclear matrix proteins were analyzed by highresolution twodimensional gel electrophoresis and computerassisted image analysis Results As2O3 significantly inhibited the growth of chronic myelogenous leukemia cell line K562 at low concentrations While more than 200 protein spots were shared among the nuclear matrices, about 18 distinct spots in the nuclear matrices were found characteristic for As2O3 treated cells Conclusions: As2O3 induces apoptosis in K562 cells in a dose and timedependent manner Our results demonstrated that for the detection of the onset of apoptosis, the alteration in the composition of nuclear matrix proteins was a more sensitive indicator than nucleosomal DNA fragmentation test These results indicated that As2O3 might be clinically useful in the treatment of chronic myelogenous leukemia. The changes of nuclear matrix proteins in the treated cells can be used as a useful indicator for this treatment展开更多
OBJECTIVE: To probe insights into the reversal effect of bufalin on vincristine-acquired multidrug resistance(MDR) in human leukemia cell line K562/VCR.METHODS: Proliferative inhibition rate and the reversal index(RI)...OBJECTIVE: To probe insights into the reversal effect of bufalin on vincristine-acquired multidrug resistance(MDR) in human leukemia cell line K562/VCR.METHODS: Proliferative inhibition rate and the reversal index(RI) of bufalin were determined by Methyl thiazolyl tetrazolium assay. The uptake of Adriamycin(ADM) in K562/VCR cells, cell cycle and apoptosis rate were determined by flow cytometry(FCM). Cell morphologic changes were observed with Wright-Giemsa staining. The expression of P-glycoprotein(P-gp), multidrug-associated protein-1(MRP1), Bcl-x L and Bax protein were measured by immunocytochemistry.RESULTS: The human leukemia multidrug resistant K562/VCR cells showed no cross-resistance to bufalin. The RIs of bufalin at concentrations of 0.0002,0.001 and 0.005 μmol/L were 4.85, 6.94 and 14.77,respectively. Preincubation of 0.001 μmol/L bufalin for 2 h could increase intracellular ADM fluorescence intensity to 28.07%(P<0.05) and down-regulate MRP1 expression simultaneously, but no remarkable effect was found on P-gp protein. Cell cycle analysis indicated increased apoptosis rate and apparent decreased G2/M phase proportion after treatment with bufalin. When exposed to 0.01μmol/L bufalin, typical morphological changes of apoptosis could be observed. Down-regulation of Bcl-x L and up-regulation of Bax expression in K562/VCR cells could be detected by immunocytochemistry.CONCLUSION: Bufalin could partly reverse the MDR of K562/VCR cells, with a possible mechanism of down-regulating MRP1 expression and activating apoptosis pathway by altering Bcl-x L/Bax ratio.展开更多
AIM: To review the characteristics of hematological malignancies in tropical areas, and to focus on the specific difficulties regarding their management. METHODS: This is a retrospective narrative review of cases of p...AIM: To review the characteristics of hematological malignancies in tropical areas, and to focus on the specific difficulties regarding their management. METHODS: This is a retrospective narrative review of cases of patients with hematological malignancies. All medical files of patients with malignant disease whose treatment was coordinated by the HematoOncology service of the Cayenne Hospital in French Guiana between the 1st of January 2010 and the 31 st of December 2012 were reviewed. Clinical data were extracted from the medical files and included: Demographic data, comorbidities, serological status for human immunodeficiency virus, human T-lymphotropic virus 1(HTLV1), hepatitis B virus and hepatitis C virusinfections, cytology and pathology diagnoses, disease extension, treatment, organization of disease management, and follow-up. The subgroup of patients with hematological malignancies and virus-related malignancies were reviewed. Cases involving patients with Kaposi sarcoma, and information on solid tumor occurrence in virus-infected patients in the whole patient population were included. Since the data were rendered anonymous, no informed consent was obtained from the patients for this retrospective analysis. Data were compiled using EXCEL® software, and the data presentation is descriptive only. The references search was guided by the nature of the data and discussion. RESULTS: In total, the clinical files of 594 patients(pts) were reviewed. Hematological malignancies were observed in 87 patients, and Kaposi sarcoma in 2 patients. In total, 70 patients had a viral infection, and 34 of these also had hematological malignancies. The hematological diagnoses were: Multiple myeloma in 27 pts, lymphoma(L) in 43 pts, myeloproliferative disorders in 17 pts and Kaposi sarcoma in two patients. The spectrum of non-Hodgkin lymphomas(NHL) was: Burkitt L(1 pt), follicular L(5 pts), chronic lymphocytic leukemia(5 pts), high-grade NHL(9 pts), mucosa-associated lymphoid tissue NHL(4 pts), T-cell lymphoma(4 pts), Adult T-cell lymphoma-leukemia(ATL)/lymphoma/leukemia(12 pts); three patients had Hodgkin disease. The spectrum of myeloproliferative diseases was: Chronic myelogenous leukemia(8 pts), thrombocytemia(5 pts) and acute leukemia(4 pts). There were no polycythemia vera, myelosclerosis, and myelodysplastic diseases. This appears to be due to bias in the recruitment process. The most important observations were: The specificity of HTLV1- related ATL malignancies, and the high incidence of virus infections in patients with hematological malignancies. Further, we noted several limitations regarding the treatment and organization of disease management. These were not related to the health care organization, but were due to a lack of board-certified hematooncology specialists, a lack of access to diagnostic tools(e.g., cytogenetic and molecular diagnosis, imaging techniques), the unavailability of radiotherapy, and the physical distance from mainland France. Yet the geography and cultures of the country also contributed to the encountered difficulties. These same limitations are seen in tropical countries with low and intermediate household incomes, but they are amplified by economic, social, and cultural issues. Thus, there is often little access to diagnostic procedures, adequate clinical management, and an unavailability of suitable medical treatments. Programs have been developed to establish centers of excellence, training in pathology diagnosis, and to provide free access to treatment.CONCLUSION: Management of hematological malignancies in tropical areas requires particular skills regarding specific features of these diseases and in terms of the affected populations, as well as solid public health policies.展开更多
基金ThisresearchwassupportedbyagrantfromtheShannxiProvincialScienceFoundationofPublicHealthBureau (No .0 0 12 2 )
文摘OBJECTIVE: To establish a serum-free culture system of dendritic cells (DCs) from chronic myeloid leukemia (CML) cells so that DCs vaccine may be applied to the adoptive immunotherapy of CML in the near future. METHODS: Fetal calf serum, serum-free medium and autologous serum were used for culture of DCs. The usage of cytokines was classified into two groups: group A (stem cell factor, granulocyte/macrophage colony-stimulating-factor, tumor necrosis factor-alpha and interleukin-4) and group B (granulocyte/macrophage colony-stimulating-factor, tumor necrosis factor-alpha and interleukin-4). The phenotypes of DCs were analyzed by using indirect immunofluorescence and flow cytometry. Mixed leukocyte responses were performed by methyl thiazolyl tetrazolium (MTT) assay. Chromosome analysis of DCs can be achieved by displaying G banding. T cells from CML patients were stimulated with autologous DCs and T-cell cytotoxicity was measured by (MTT) assay. RESULTS: CD34(+) cells or mononuclear cells were obtained from peripheral blood or bone marrow samples of eight patients of chronic-phase CML. Group A of serum-free medium was better than group B in expansion of total cell numbers and the rate of DCs. These results of serum-free medium were not significantly different from those of fetal calf serum medium, but the results of autologous serum medium were inferior to two groups above. The expression of major histocompatibility complex class II antigen on the surface of DCs was notable (> 50%), but the expression of CD83 and the costimulatory molecules CD86 was not noticeable (10% - 50%). Although CD1a(+)/CD14(-) DCs were potent stimulators of allogeneic lymphocytes, expansion of T cells from normal volunteers were not significant (average 27.2 fold at DCs: T cells ratio of 1:10). At day 12, CD1a(+) cells from three patients were studied by displaying G banding and Ph(+) cells in these populations were 100%, 98% and 60%, respectively. At an effector: target ratio of 40:1, 32% to 45% cytotoxicity was noted with DC-stimulated T cells against autologous leukemia cells. CONCLUSIONS: A stable serum-free culture system of CML-DCs was established. The expression of CD83 and CD86 on the surface of CML-DCs and DCs' potent stimulation of allogeneic lymphocytes were not notable. DCs in CML patients can be derived from the malignant clone and these malignant DCs could induce anti-leukemic reactivity in autologous T lymphocytes without the necessity for additional exogenous antigens.
文摘OBJECTIVE To investigate the effects of survivin antisense oligodeoxy-nucleotid (ASODN) on proliferation and apoptosis in the chronic myeloid leukemia cell line K562. METHODS Different concentrations of an antisense oligodeoxy-nucleotid and control sequence (scrambled ODN) targeting the survivin gene were transferred into K562 by a lipofectin reagent. The MTT assay was used to measure the growth inhibitory rate, IC50, and to observe the cytotoxicity of survivin ASODN in the K562 cells. The morphologic changes in the nucleus and the apoptotic rate were observed by Hoechst33342/PI staining. Caspase-3 activity was evaluated by a kinase activity assay. The changes of survivin protein expression after transfection were detected by Western blots. RESULTS Eight hours after transfection, fluorescence in the K562 cells was well distributed. Treatment of the cells for 44 h with different concentrations of survivin ASODN produced a IC50 of 800 nmol/L. The growth inhibitory rate with 200, 400, 600 and 1000 nmol/L of survivin ASODN was 15.8±1.6%, 23.8±5.9%, 37.1±5.6% and 77.3±2.5% respectively. After 36 h of of survivin ASODN treatment, distinct morphologic changes characteristic of cell apoptosis such as karyopyknosis and conglomeration were observed by Hoechst33342/PI staining. Caspase-3 activity increased significantly after treatment of the cells with different concentrations of survivin ASODN(P<0.01)and following treatment with 800 nmol/L survivin ASODN, survivin expression decreased significantly. CONCLUSION Survivin ASODN exerts an anti-cancer effect by inducing apoptosis in K562 leukaemia cells. Up-regulated expression of caspase-3 may play a role in this process.
文摘Background Arsenic trioxide (As2O3) has been identified as a very potent antiacute leukemic agent However its role in apoptosis needs to be elucidated As2O3 interferes with the proliferation and survival of tumor cells via a variety of mechanisms Drugtarget interactions at the level of nuclear matrix (NM) may be critical events in the induction of cell death by As2O3 This study dealt with As2O3-target interactions at the level of NM in chronic myelogenous leukemia cell line K562 by proteomics Methods K562 cells were cultured in MEM and treated with different concentrations of As2O3 The nuclear matrix proteins were analyzed by highresolution twodimensional gel electrophoresis and computerassisted image analysis Results As2O3 significantly inhibited the growth of chronic myelogenous leukemia cell line K562 at low concentrations While more than 200 protein spots were shared among the nuclear matrices, about 18 distinct spots in the nuclear matrices were found characteristic for As2O3 treated cells Conclusions: As2O3 induces apoptosis in K562 cells in a dose and timedependent manner Our results demonstrated that for the detection of the onset of apoptosis, the alteration in the composition of nuclear matrix proteins was a more sensitive indicator than nucleosomal DNA fragmentation test These results indicated that As2O3 might be clinically useful in the treatment of chronic myelogenous leukemia. The changes of nuclear matrix proteins in the treated cells can be used as a useful indicator for this treatment
基金Shanghai Municipal Health Bureau:Traditional Chinese Medicine in Treating with Advanced Hepatocellular Carcinoma(No.ZYSNXD-CC-ZDYJ032)
文摘OBJECTIVE: To probe insights into the reversal effect of bufalin on vincristine-acquired multidrug resistance(MDR) in human leukemia cell line K562/VCR.METHODS: Proliferative inhibition rate and the reversal index(RI) of bufalin were determined by Methyl thiazolyl tetrazolium assay. The uptake of Adriamycin(ADM) in K562/VCR cells, cell cycle and apoptosis rate were determined by flow cytometry(FCM). Cell morphologic changes were observed with Wright-Giemsa staining. The expression of P-glycoprotein(P-gp), multidrug-associated protein-1(MRP1), Bcl-x L and Bax protein were measured by immunocytochemistry.RESULTS: The human leukemia multidrug resistant K562/VCR cells showed no cross-resistance to bufalin. The RIs of bufalin at concentrations of 0.0002,0.001 and 0.005 μmol/L were 4.85, 6.94 and 14.77,respectively. Preincubation of 0.001 μmol/L bufalin for 2 h could increase intracellular ADM fluorescence intensity to 28.07%(P<0.05) and down-regulate MRP1 expression simultaneously, but no remarkable effect was found on P-gp protein. Cell cycle analysis indicated increased apoptosis rate and apparent decreased G2/M phase proportion after treatment with bufalin. When exposed to 0.01μmol/L bufalin, typical morphological changes of apoptosis could be observed. Down-regulation of Bcl-x L and up-regulation of Bax expression in K562/VCR cells could be detected by immunocytochemistry.CONCLUSION: Bufalin could partly reverse the MDR of K562/VCR cells, with a possible mechanism of down-regulating MRP1 expression and activating apoptosis pathway by altering Bcl-x L/Bax ratio.
文摘AIM: To review the characteristics of hematological malignancies in tropical areas, and to focus on the specific difficulties regarding their management. METHODS: This is a retrospective narrative review of cases of patients with hematological malignancies. All medical files of patients with malignant disease whose treatment was coordinated by the HematoOncology service of the Cayenne Hospital in French Guiana between the 1st of January 2010 and the 31 st of December 2012 were reviewed. Clinical data were extracted from the medical files and included: Demographic data, comorbidities, serological status for human immunodeficiency virus, human T-lymphotropic virus 1(HTLV1), hepatitis B virus and hepatitis C virusinfections, cytology and pathology diagnoses, disease extension, treatment, organization of disease management, and follow-up. The subgroup of patients with hematological malignancies and virus-related malignancies were reviewed. Cases involving patients with Kaposi sarcoma, and information on solid tumor occurrence in virus-infected patients in the whole patient population were included. Since the data were rendered anonymous, no informed consent was obtained from the patients for this retrospective analysis. Data were compiled using EXCEL® software, and the data presentation is descriptive only. The references search was guided by the nature of the data and discussion. RESULTS: In total, the clinical files of 594 patients(pts) were reviewed. Hematological malignancies were observed in 87 patients, and Kaposi sarcoma in 2 patients. In total, 70 patients had a viral infection, and 34 of these also had hematological malignancies. The hematological diagnoses were: Multiple myeloma in 27 pts, lymphoma(L) in 43 pts, myeloproliferative disorders in 17 pts and Kaposi sarcoma in two patients. The spectrum of non-Hodgkin lymphomas(NHL) was: Burkitt L(1 pt), follicular L(5 pts), chronic lymphocytic leukemia(5 pts), high-grade NHL(9 pts), mucosa-associated lymphoid tissue NHL(4 pts), T-cell lymphoma(4 pts), Adult T-cell lymphoma-leukemia(ATL)/lymphoma/leukemia(12 pts); three patients had Hodgkin disease. The spectrum of myeloproliferative diseases was: Chronic myelogenous leukemia(8 pts), thrombocytemia(5 pts) and acute leukemia(4 pts). There were no polycythemia vera, myelosclerosis, and myelodysplastic diseases. This appears to be due to bias in the recruitment process. The most important observations were: The specificity of HTLV1- related ATL malignancies, and the high incidence of virus infections in patients with hematological malignancies. Further, we noted several limitations regarding the treatment and organization of disease management. These were not related to the health care organization, but were due to a lack of board-certified hematooncology specialists, a lack of access to diagnostic tools(e.g., cytogenetic and molecular diagnosis, imaging techniques), the unavailability of radiotherapy, and the physical distance from mainland France. Yet the geography and cultures of the country also contributed to the encountered difficulties. These same limitations are seen in tropical countries with low and intermediate household incomes, but they are amplified by economic, social, and cultural issues. Thus, there is often little access to diagnostic procedures, adequate clinical management, and an unavailability of suitable medical treatments. Programs have been developed to establish centers of excellence, training in pathology diagnosis, and to provide free access to treatment.CONCLUSION: Management of hematological malignancies in tropical areas requires particular skills regarding specific features of these diseases and in terms of the affected populations, as well as solid public health policies.
