Generation of Reactive Oxygen Species during Apoptosis Induced by DNA-Damaging Agents and/or Histone Deacetylase Inhibitors

Reactive oxygen species play an important role in the process of apoptosis in many cell types. In this paper, we analyzed the role of ROS in DNA-damaging agents (actinomycin D or decitabine), which induced apoptosis of leukemia cell line CML-T1 and normal peripheral blood lymphocytes (PBL). The possibility of synergism with histone deacetylase inhibitors butyrate or SAHA is also reported. We found that in cancer cell line, ROS production significantly contributed to apoptosis triggering, while in normal lymphocytes treated by cytostatic or cytotoxic drugs, necrosis as well as apoptosis occurred and large heterogeneity of ROS production was measured. Combined treatment with histone deacetylase inhibitor did not potentiate actinomycin D action, whereas combination of decitabine and SAHA brought synergistic ROS generation and apoptotic features in CML cell line. Appropriate decrease of cell viability indicated promising therapeutic potential of this combination in CML, but side effects on normal PBL should be taken into attention

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Katedra výtvarné výchovyPedagogická fakultaFaculty of Educatio

The Legislative Approach of Countries to Abortion from the Perspective of Cultural Dimensions in the International Comparison

Studium humanitní vzdělanosti - Společenskovědní modulLiberal Arts and Humanities - Social Sciences ModuleFaculty of HumanitiesFakulta humanitních studi

Comparison of Karmapas statements by critical discourse analysis in the light of postmaterialistic values concept defined by Inglehard

This dissertation is mainly focused on schism in the Karma-kagjü school. The death of the 16th Karmapa Rangjung Rigpe Dorje († 1981), the head of Kagjüpa plunged Karma-kagjü school to inner struggle for power and prestige. Enmity was making progress slowly and hiddenly. Situation graded in clandestine fashion and culminated in 1992 when Ogyen Trinley Dorje was enthroned in Tibet as 17th Karmapa. Some of the regents had doubt about him being the Karmapa and consequently enthroned their own candidate for 17th Karmapa. This act entirely divided the Karma-kagjü school. The primary motive and goal of my dissertation is to find presence of materialistic or postmaterialistic values defined by Ronald Inglehart (Inglehart 1997) in interviews given by 17th Karmapas. I'm looking for difference in values and my methodical instrument is critical discourse analysis. After comparison of interviews of both 17th Karmapas I find the difference in their values. My conclusion is that when Ogyen Trinley Dorje has more materialistic values than Trinley Thaye Dorje. This dissertation contains history of Tibet, history of Buddhism in Tibet and foreign relation interference in practice of Buddhism in Tibet (China and Mongolia). In addition I describe political situation of Tibet and Tibetan exile government which interfere in..

Chemotherapy-Induced Survivin Regulation in Acute Myeloid Leukemia Cells

Survivin is a 16.5 kDa protein highly expressed in centrosomes, where it controls proper sister chromatid separation. In addition to its function in mitosis, survivin is also involved in apoptosis. Overexpression of survivin in many cancer types makes it a suitable target for cancer therapy. Western blotting and confocal microscopy were used to characterize the effect of chemotherapy on acute myeloid leukemia (AML) cells. We found enhanced survivin expression in a panel of AML cell lines treated with cytarabine (Ara-C), which is part of a first-line induction regimen for AML therapy. Simultaneously, Ara-C caused growth arrest and depletion of the mitotic cell fraction. Subsequently, the effect of a second component of standard therapy protocol, idarubicin, and of a known survivin inhibitor, YM-155, on cell viability and survivin expression and localization in AML cells was investigated. Idarubicin reversed Ara-C-induced survivin upregulation in the majority of AML cell lines. YM-155 caused survivin deregulation together with a viability decrease in cells resistant to idarubicin treatment, suggesting that YM-155 might be efficient in a specific subset of AML patients. Expression levels of other apoptosis-related proteins, in particular X-linked inhibitor of apoptosis (XIAP), Mcl-1, and p53, and of the cell-cycle inhibitor p21 considerably changed in almost all cases, confirming the off-target effects of YM-155

Decitabine and SAHA-Induced Apoptosis Is Accompanied by Survivin Downregulation and Potentiated by ATRA in p53-Deficient Cells

While p53-dependent apoptosis is triggered by combination of methyltransferase inhibitor decitabine (DAC) and histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA) in leukemic cell line CML-T1, reactive oxygen species (ROS) generation as well as survivin and Bcl-2 deregulation participated in DAC + SAHA-induced apoptosis in p53-deficient HL-60 cell line. Moreover, decrease of survivin expression level is accompanied by its delocalization from centromere-related position in mitotic cells suggesting that both antiapoptotic and cell cycle regulation roles of survivin are affected by DAC + SAHA action. Addition of subtoxic concentration of all-trans-retinoic acid (ATRA) increases the efficiency of DAC + SAHA combination on viability, apoptosis induction, and ROS generation in HL-60 cells but has no effect in CML-T1 cell line. Peripheral blood lymphocytes from healthy donors showed no damage induced by DAC + SAHA + ATRA combination. Therefore, combination of ATRA with DAC and SAHA represents promising tool for therapy of leukemic disease with nonfunctional p53 signalization

Localization of AML-related nucleophosmin mutant depends on its subtype and is highly affected by its interaction with wild-type NPM.

Mutations of the gene for nucleophosmin (NPM1) are the most frequent genetic aberration in patients with acute myeloid leukemia (AML). The mechanism of leukemic transformation in this leukemia subtype is not fully understood, but aberrant cytoplasmic localization of mutated NPM (NPMmut) is widely considered as an important factor for leukemia manifestation. We analyzed the subcellular localization of three types of NPM with a C-terminal mutation (A, B and E). Genes for the individual NPM forms were fused with a gene for one of fluorescent protein variants in plasmids, which were transfected into three cell lines with different endogenous NPM expression. Subcellular localization of the fluorescent protein-labeled NPM was further correlated with the relative expression of all NPM forms. We confirmed a high cytoplasmic expression of NPMmutA and NPMmutB whereas a substantial fraction of NPMmutE was found to be localized in nucleoli. Moreover, we revealed that the localization of fluorescently labeled NPM is affected by the interaction between various forms of the protein

Combined Treatment with Low Concentrations of Decitabine and SAHA Causes Cell Death in Leukemic Cell Lines but Not in Normal Peripheral Blood Lymphocytes

Epigenetic therapy reverting aberrant acetylation or methylation offers the possibility to target preferentially tumor cells and to preserve normal cells. Combination epigenetic therapy may further improve the effect of individual drugs. We investigated combined action of demethylating agent decitabine and histone deacetylase inhibitor SAHA (Vorinostat) on different leukemic cell lines in comparison with peripheral blood lymphocytes. Large decrease of viability, as well as huge p21WAF1 induction, reactive oxygen species formation, and apoptotic features due to combined decitabine and SAHA action were detected in leukemic cell lines irrespective of their p53 status, while essentially no effect was observed in response to the combined drug action in normal peripheral blood lymphocytes of healthy donors. p53-dependent apoptotic pathway was demonstrated to participate in the wtp53 CML-T1 leukemic cell line response, while significant influence of reactive oxygen species on viability decrease has been detected in p53-null HL-60 cell line