Objective： To investigate the effects of soluble factors secreted by acute myeloid leukemia （AML） cells on the phenotypical and functional properties of DCs derived from normal mononuclear cells. Methods： Mononucl...Objective： To investigate the effects of soluble factors secreted by acute myeloid leukemia （AML） cells on the phenotypical and functional properties of DCs derived from normal mononuclear cells. Methods： Mononuclear cells were cultured with interleukin-4 （IL-4） and granulocyte-macrophage colony-stimulating factor （GM-CSF）, in the presence or absence of 24 h culture supematants from fresh pdmary AML cells, to generate immature DCs. The maturation of DCs was induced by cytokines IL-lbeta, IL-6, tumor necrosis factor-alpha （TNF-alpha）, and prostaglandin-2 （PGE-2）. The phenotypic alterations of DCs and DCs-primed CD4＋ T cells were evaluated using flow cytometry. Precursor frequency （PF） was calculated to monitor the allostimulatory effects of DCs on CD4^＋ and CD8^＋ T cells. Results：AML cell supernatant-treated DCs showed significantly lower expression of co-stimulatory molecules CDS0 and CD86, and reduced response to cytokines IL-1beta, IL-6, TNF-alpha, and PGE-2. The allostimulatory effects of AML cell supematant-treated DCs on CD4^＋ and CD8^＋ T cells were significantly lower than those of normal mature DCs [PF： （1.8 ±0.5）% vs. （5.2 ± 1.6）% for CD4^＋ T cells, （2.1 ±0.6）% vs. （6.5 ± 2.0）% for CD8^＋ T cells, P 〈 0.01]. These AML supernatantoinduced DCs could also induce allogeneic CD4^＋ T cells to differentiate into CD4^＋CD25high T cells, which had immunophenotyping characteristics of regulatory T cells, i.e. they expressed Foxp3 but not active maker CD69. Conclusion： This study demonstrates that soluble factors secreted by AML cells can inhibit development and functions of DCs. In addition, AML supernatant-induced DCs can induce the generation of CD4^＋CD25^high T cells from CD4^＋ T cells, which may be a mechanism of increased prevalence of CD4^＋CD25^high regulatory T cells and immune dysfunction in AML patients.展开更多
This study examined the signaling events induced by shikonin that lead to the induction of apoptosis in Bcr/ Abl-positive chronic myelogenous leukemia （CML） cells （e.g., K562, LAMA84）. Treatment of K562 cells with...This study examined the signaling events induced by shikonin that lead to the induction of apoptosis in Bcr/ Abl-positive chronic myelogenous leukemia （CML） cells （e.g., K562, LAMA84）. Treatment of K562 cells with shikonin （e.g., 0.5 pM） resulted in profound induction of apoptosis accompanied by rapid generation of reactive oxygen species （ROS）, striking activation of c-Jun-N-terminal kinase （JNK） and p38, marked release of the mitochondrial proteins cytochrome c and Smac/DIABLO, activation of caspase-9 and -3, and cleavage of PARP. Scavenging of ROS completely blocked all of the above-mentioned events （i.e., JNK and p38 phosphorylation, cytochrome c and Smac/DIABLO release, caspase and PARP cleavage, as well as the induction of apoptosis） following shikonin treatment. Inhibition of JNK and knock-down of JNK1 significantly attenuated cytochrome c release, caspase cleavage and apoptosis, but did not affect shikonin-mediated ROS production. Additionally, inhibition of caspase activation completely blocked shikonin-induced apoptosis, but did not appreciably modify shikonin-mediated cytochrome c release or ROS generation. Altogether, these findings demonstrate that shikonin-induced oxidative injury operates at a proximal point in apoptotic signaling cascades, and subsequently activates the stress-related JNK pathway, triggers mitochondrial dysfunction, cytochrome c release, and caspase activation, and leads to apoptosis. Our data also suggest that shikonin may be a promising agent for the treatment of CML, as a generator of ROS.展开更多
基金grants from the National Outstanding Young Investigator Program (30225038)Anhui Provincial Natural Science Foundation (070413094)+1 种基金Scientific Research Fund of Anhui Provincial Education Department (2006KJ072C)Science and Technological Fund of Anhui Province for Outstanding Youth.
文摘Objective： To investigate the effects of soluble factors secreted by acute myeloid leukemia （AML） cells on the phenotypical and functional properties of DCs derived from normal mononuclear cells. Methods： Mononuclear cells were cultured with interleukin-4 （IL-4） and granulocyte-macrophage colony-stimulating factor （GM-CSF）, in the presence or absence of 24 h culture supematants from fresh pdmary AML cells, to generate immature DCs. The maturation of DCs was induced by cytokines IL-lbeta, IL-6, tumor necrosis factor-alpha （TNF-alpha）, and prostaglandin-2 （PGE-2）. The phenotypic alterations of DCs and DCs-primed CD4＋ T cells were evaluated using flow cytometry. Precursor frequency （PF） was calculated to monitor the allostimulatory effects of DCs on CD4^＋ and CD8^＋ T cells. Results：AML cell supernatant-treated DCs showed significantly lower expression of co-stimulatory molecules CDS0 and CD86, and reduced response to cytokines IL-1beta, IL-6, TNF-alpha, and PGE-2. The allostimulatory effects of AML cell supematant-treated DCs on CD4^＋ and CD8^＋ T cells were significantly lower than those of normal mature DCs [PF： （1.8 ±0.5）% vs. （5.2 ± 1.6）% for CD4^＋ T cells, （2.1 ±0.6）% vs. （6.5 ± 2.0）% for CD8^＋ T cells, P 〈 0.01]. These AML supernatantoinduced DCs could also induce allogeneic CD4^＋ T cells to differentiate into CD4^＋CD25high T cells, which had immunophenotyping characteristics of regulatory T cells, i.e. they expressed Foxp3 but not active maker CD69. Conclusion： This study demonstrates that soluble factors secreted by AML cells can inhibit development and functions of DCs. In addition, AML supernatant-induced DCs can induce the generation of CD4^＋CD25^high T cells from CD4^＋ T cells, which may be a mechanism of increased prevalence of CD4^＋CD25^high regulatory T cells and immune dysfunction in AML patients.
基金Acknowledgments This work was supported by grants from the National High Technology Research and Development Program of China （863 Program） （2006AA02A306）, the National Natural Science Foundation of China （No. 39900082） and the PhD Program Foundation of Ministry of Education of China （No. 204090188）. We thank Dr Courtney M Heney （Massachusetts General Hospital, Harvard University） for critically reading the manuscript.
文摘This study examined the signaling events induced by shikonin that lead to the induction of apoptosis in Bcr/ Abl-positive chronic myelogenous leukemia （CML） cells （e.g., K562, LAMA84）. Treatment of K562 cells with shikonin （e.g., 0.5 pM） resulted in profound induction of apoptosis accompanied by rapid generation of reactive oxygen species （ROS）, striking activation of c-Jun-N-terminal kinase （JNK） and p38, marked release of the mitochondrial proteins cytochrome c and Smac/DIABLO, activation of caspase-9 and -3, and cleavage of PARP. Scavenging of ROS completely blocked all of the above-mentioned events （i.e., JNK and p38 phosphorylation, cytochrome c and Smac/DIABLO release, caspase and PARP cleavage, as well as the induction of apoptosis） following shikonin treatment. Inhibition of JNK and knock-down of JNK1 significantly attenuated cytochrome c release, caspase cleavage and apoptosis, but did not affect shikonin-mediated ROS production. Additionally, inhibition of caspase activation completely blocked shikonin-induced apoptosis, but did not appreciably modify shikonin-mediated cytochrome c release or ROS generation. Altogether, these findings demonstrate that shikonin-induced oxidative injury operates at a proximal point in apoptotic signaling cascades, and subsequently activates the stress-related JNK pathway, triggers mitochondrial dysfunction, cytochrome c release, and caspase activation, and leads to apoptosis. Our data also suggest that shikonin may be a promising agent for the treatment of CML, as a generator of ROS.
