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

ixFLIM: Interferometric Excitation Fluorescence Lifetime Imaging Microscopy

Fluorescence lifetime imaging microscopy (FLIM) is a well-established technique with numerous imaging applications. Yet, one of the limitations of FLIM is that it provides information about the emitting state only. Here, we present an extension of FLIM by interferometric measurement of fluorescence excitation spectra. Interferometric Excitation Fluorescence Lifetime Imaging Microscopy (ixFLIM) reports on the correlation of the excitation spectra and emission lifetime, providing the correlation between the ground-state absorption and excited-state emission. As such, it extends the applicability of FLIM and removes some of its limitations. We introduce ixFLIM on progressively more complex systems and apply it to quantitative resonance energy transfer imaging from a single measurement.Comment: 29 pages (19 main and 10 supporting), 18 figure

AML-Related NPM Mutations Drive p53 Delocalization into the Cytoplasm with Possible Impact on p53-Dependent Stress Response

Nucleophosmin (NPM) interaction with tumor suppressor p53 is a part of a complex interaction network and considerably affects cellular stress response. The impact of NPM1 mutations on its interaction with p53 has not been investigated yet, although consequences of NPMmut-induced p53 export to the cytoplasm are important for understanding the oncogenic potential of these mutations. We investigated p53-NPM interaction in live HEK-293T cells by FLIM-FRET and in cell lysates by immunoprecipitation. eGFP lifetime-photoconversion was used to follow redistribution dynamics of NPMmut and p53 in Selinexor-treated cells. We confirmed the p53-NPMwt interaction in intact cells and newly documented that this interaction is not compromised by the NPM mutation causing displacement of p53 to the cytoplasm. Moreover, the interaction was not abolished for non-oligomerizing NPM variants with truncated oligomerization domain, suggesting that oligomerization is not essential for interaction of NPM forms with p53. Inhibition of the nuclear exporter XPO1 by Selinexor caused expected nuclear relocalization of both NPMmut and p53. However, significantly different return rates of these proteins indicate nontrivial mechanism of p53 and NPMmut cellular trafficking. The altered p53 regulation in cells expressing NPMmut offers improved understanding to help investigational strategies targeting these mutations

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

AML-Related NPM Mutations Drive p53 Delocalization into the Cytoplasm with Possible Impact on p53-Dependent Stress Response

Nucleophosmin (NPM) interaction with tumor suppressor p53 is a part of a complex interaction network and considerably affects cellular stress response. The impact of NPM1 mutations on its interaction with p53 has not been investigated yet, although consequences of NPMmut-induced p53 export to the cytoplasm are important for understanding the oncogenic potential of these mutations. We investigated p53-NPM interaction in live HEK-293T cells by FLIM-FRET and in cell lysates by immunoprecipitation. eGFP lifetime-photoconversion was used to follow redistribution dynamics of NPMmut and p53 in Selinexor-treated cells. We confirmed the p53-NPMwt interaction in intact cells and newly documented that this interaction is not compromised by the NPM mutation causing displacement of p53 to the cytoplasm. Moreover, the interaction was not abolished for non-oligomerizing NPM variants with truncated oligomerization domain, suggesting that oligomerization is not essential for interaction of NPM forms with p53. Inhibition of the nuclear exporter XPO1 by Selinexor caused expected nuclear relocalization of both NPMmut and p53. However, significantly different return rates of these proteins indicate nontrivial mechanism of p53 and NPMmut cellular trafficking. The altered p53 regulation in cells expressing NPMmut offers improved understanding to help investigational strategies targeting these mutations

NPM is localized in the cytoplasm of blast from AML patients with NPM mutation.

<p>PBMC from AML patients with NPMwt (wt), NPMmutA (mut A) or NPMmutNm (mut Nm) were incubated with anti-NPM (clone 3F291) primary and AlexaFluor555 secondary antibodies (red). The nuclei were visualized with Hoechst 33342 (blue). Arrows indicate the cytoplasmic localization of NPM in AML blasts with NPMmut. The bars represent 10μm.</p

Interaction between wild-type and mutant affects localization of individual forms of NPM.

<p>(a) eGFP (green) and mRFP1 (red) fluorescence from HEK-293T cells co-transfected with mRFP1_NPMwt and eGFP_NPMmutA (mutA) or eGFP_NPMmutE (mutE). The bars represent 20 μm. (b) fraction of transfected cells displaying eGFP_NPM signal only from the cytoplasm (white bars), from the cytoplasm and nucleoli (grey bars) or only from nucleoli (black bar). GFP_mut denotes the signal from cells transfected with eGFP_NPMmut only, +RFP_wt denotes eGFP signal from cells co-transfected with eGFP_NPMmut and mRFP1_NPMwt. The error bars in the graph represent ±SD of at least 3 independent experiments. (c) fraction of transfected cells displaying mRFP1_NPMwt signal from the cytoplasm: wt—cells transfected only with RFP_NPMwt, wt+mutA (or E)–cells co-transfected with RFP_NPMwt and GFP_NPMmutA (or E). The error bars in the graph represent ±SD of 5 independent experiments. Statistical significance degree of difference between the samples: P < 0.01 (**), P < 0.001 (***).</p