Reactivating p53 and Inducing Tumor Apoptosis (RITA) has been reported to increase the p53 activity and to trigger p53-dependent apoptosis in cancer cells with wild-type p53. Tumor suppressor p53 interacts with nucleolar phosphoproteins nucleophosmin (NPM) and nucleolin (NCL), which have crucial role in many cellular processes. Specific NPM mutations associated with acute myeloid leukemia (AML) cause aberrant localization of NPM and p53 in the cytoplasm with possible impact on the p53 function. We tested an effect of RITA on primary cells, and we found significant RITA-induced changes in NPM and NCL phosphorylation associated with apoptosis in cells of AML patients, but not that of healthy donors. Subsequent screening of several AML cell lines revealed heterogeneous response to RITA, and confirmed an association of the specific phosphorylation with apoptosis. While decreased NCL phosphorylation at Threonines T76 and T84 could be attributed to RITA-induced cell cycle arrest, enhanced NPM phosphorylation at Threonine T199 was not accompanied by the cell cycle changes and it correlated with sensitivity to RITA. Simultaneously, inverse changes occurred at Serine S4 of the NPM. These new findings of RITA mechanism of action could establish the NPM pT199/pS4 ratio as a marker for suitability of RITA treatment of AML cells.

• Klíčová slova
• Acute myeloid leukemia, Apoptosis, Nucleolin, Nucleophosmin, Phosphorylation, RITA, p53,
• MeSH
• akutní myeloidní leukemie * metabolismus   MeSH
• apoptóza MeSH
• buněčné jadérko metabolismus   MeSH
• fosfoproteiny genetika   metabolismus   MeSH
• lidé MeSH
• nádorový supresorový protein p53 * genetika   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• fosfoproteiny MeSH
• nádorový supresorový protein p53 * MeSH

P21-activated kinases (PAK) regulate processes associated with cytoskeleton dynamics. PAK expression in leukemia cells was measured on protein and mRNA levels. In functional assays, we analyzed the effect of PAK inhibitors IPA-3 and FRAX597 on cell adhesivity and viability. PAK2 was dominant in cell lines, whereas primary cells also expressed comparable amount of PAK1 transcription isoforms: PAK1-full and PAK1Δ15. PAK1Δ15 and PAK2 levels correlated with surface density of integrins β1 and αVβ3. PAK1-full, but not PAK2, was present in membrane protrusions. IPA-3, which prevents PAK activation, induced cell contraction in semi-adherent HEL cells only. FRAX597, which inhibits PAK kinase activity, increased cell-surface contact area in all leukemia cells. Both inhibitors reduced the stability of cell attachment and induced cell death.

• Klíčová slova
• AML, ECIS, IRM, PAK, acute myeloid leukemia, cell adhesion,
• MeSH
• buněčná adheze MeSH
• buněčné linie MeSH
• fibronektiny genetika   MeSH
• leukemie * genetika   MeSH
• lidé MeSH
• p21 aktivované kinasy * genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• fibronektiny MeSH
• p21 aktivované kinasy * MeSH

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.

• Klíčová slova
• FLIM-FRET, Selinexor, acute myeloid leukemia, mutation, nucleophosmin, p53, photoconversion,
• Publikační typ
• časopisecké články MeSH

Nucleophosmin (NPM) mutations causing its export from the nucleoli to the cytoplasm are frequent in acute myeloid leukemia (AML). Due to heterooligomerization of wild type NPM with the AML-related mutant, the wild-type becomes misplaced from the nucleoli and its functions are significantly altered. Dissociation of NPM heterooligomers may thus restore the proper localization and function of wild-type NPM. NSC348884 is supposed to act as a potent inhibitor of NPM oligomerization. The effect of NSC348884 on the NPM oligomerization was thoroughly examined by fluorescence lifetime imaging with utilization of FRET and by a set of immunoprecipitation and electrophoretic methods. Leukemia-derived cell lines and primary AML cells as well as cells transfected with fluorescently labeled NPM forms were investigated. Our results clearly demonstrate that NSC348884 does not inhibit formation of NPM oligomers neither in vivo nor in vitro. Instead, we document that NSC348884 cytotoxicity is rather associated with modified cell adhesion signaling. The cytotoxic mechanism of NSC348884 has therefore to be reconsidered.

• MeSH
• antitumorózní látky farmakologie   MeSH
• apoptóza účinky léků   MeSH
• HEK293 buňky MeSH
• indoly farmakologie   MeSH
• jaderné proteiny genetika   metabolismus   MeSH
• leukemie farmakoterapie   genetika   metabolismus   MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• nukleofosmin MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antitumorózní látky MeSH
• indoly MeSH
• jaderné proteiny MeSH
• NPM1 protein, human MeSH Prohlížeč
• NSC 348884 MeSH Prohlížeč
• nukleofosmin MeSH

Compared to solid tumors, the role of PD-L1 in hematological malignancies is less explored, and the knowledge in this area is mostly limited to lymphomas. However, several studies indicated that PD-L1 is also overexpressed in myeloid malignancies. Successful treatment of the acute myeloid leukemia (AML) is likely associated with elimination of the residual disease by the immune system, and possible involvement of PD-L1 in this process remains to be elucidated. We analyzed PD-L1 expression on AML primary cells by flow cytometry and, in parallel, transcript levels were determined for the transcription variants v1 and v2. The ratio of v1/v2 cDNA correlated with the surface protein amount, and high v1/v2 levels were associated with worse overall survival (p = 0.0045). The prognostic impact of PD-L1 was limited to AML with mutated nucleophosmin and concomitant internal tandem duplications in the FLT3 gene (p less than 0.0001 for this particular AML subgroup).

• Klíčová slova
• AML, CD34, FLT3-ITD, NPM1, PD-1, PD-L1 transcript, leukemia,
• MeSH
• akutní myeloidní leukemie krev   genetika   MeSH
• antigeny CD274 krev   genetika   metabolismus   MeSH
• jaderné proteiny genetika   MeSH
• lidé MeSH
• messenger RNA genetika   metabolismus   MeSH
• mutace MeSH
• nádorové biomarkery krev   genetika   metabolismus   MeSH
• nukleofosmin MeSH
• tyrosinkinasa 3 podobná fms genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• antigeny CD274 MeSH
• CD274 protein, human MeSH Prohlížeč
• FLT3 protein, human MeSH Prohlížeč
• jaderné proteiny MeSH
• messenger RNA MeSH
• nádorové biomarkery MeSH
• NPM1 protein, human MeSH Prohlížeč
• nukleofosmin MeSH
• tyrosinkinasa 3 podobná fms MeSH

C-terminal mutations of the nucleolar protein nucleophosmin (NPM) are the most frequent genetic aberration detected in acute myeloid leukemia (AML) with normal karyotype. The mutations cause aberrant cytoplasmic localization of NPM and lead to loss of functions associated with NPM nucleolar localization, e.g. in ribosome biogenesis or DNA-damage repair. NPM has many interaction partners and some of them were proved to interact also with the mutated form (NPMmut) and due to this interaction thereby to be withdrawn from their site of action. We analyzed the impact of the mutation on NPM interaction with nucleolin (NCL) which is also prevalently localized into the nucleolus and cooperates with wild-type NPM (NPMwt) in many cellular processes. We revealed that the NCL-NPM complex formation is completely abolished by the mutation and that the presence/absence of the interaction is not affected by drugs causing genotoxic stress or differentiation. Deregulation resulting from changes of NCL/NPMwt ratio may contribute to leukemogenesis.

• Klíčová slova
• AML, Interaction, Mutation, Nucleolin, Nucleophosmin, Oligomerization,
• MeSH
• akutní myeloidní leukemie genetika   metabolismus   patologie   MeSH
• buněčné jadérko genetika   metabolismus   patologie   MeSH
• fosfoproteiny genetika   metabolismus   MeSH
• HEK293 buňky MeSH
• jaderné proteiny genetika   metabolismus   MeSH
• lidé MeSH
• multiproteinové komplexy genetika   metabolismus   MeSH
• mutace * MeSH
• nádorové buněčné linie MeSH
• nádorové proteiny genetika   metabolismus   MeSH
• nukleofosmin MeSH
• nukleolin MeSH
• proteiny vázající RNA genetika   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• fosfoproteiny MeSH
• jaderné proteiny MeSH
• multiproteinové komplexy MeSH
• nádorové proteiny MeSH
• NPM1 protein, human MeSH Prohlížeč
• nukleofosmin MeSH
• proteiny vázající RNA MeSH

The expression on the surface of tumor cells of ligands for the PD-1 inhibitory receptor prevents the antitumor immune response and is considered to be a negative prognostic factor in a variety of solid tumors as well as in hematologic malignancies. To determine if it were possible to analyze PD-L1 with PCR-based methods, we assessed the expression of PD-L1 in primary samples from patients with acute myeloid leukemia, in healthy donors, and in a panel of cell lines, by means of flow cytometry, RT-PCR, and Western blotting. Although the surface density of the protein was not correlated with the amount of expressed full-length mRNA, we found a statistically significant positive correlation between PD-L1 surface density and the ratio of two transcript variants (variant 1/variant 2). Our PCR-based method allows for retrospective examination of PD-L1 surface expression from frozen cDNA samples, without the need for a reference gene. Our results also suggest that variant 2, which is produced by alternative splicing, negatively regulates PD-L1 protein expression on the cell surface. In addition, PD-L1 exposition on the cell surface is clearly associated with a shift of electrophoretic mobility, observed on Western blots. This finding can explain the relatively large variability in PD-L1 apparent molecular weight reported in the literature and offers an alternate means for the assessment of PD-L1 surface expression. Cancer Immunol Res; 4(10); 815-9. ©2016 AACR.

• MeSH
• akutní myeloidní leukemie genetika   imunologie   MeSH
• alternativní sestřih MeSH
• antigeny CD274 biosyntéza   krev   genetika   MeSH
• genetická variace MeSH
• lidé MeSH
• messenger RNA genetika   MeSH
• nádorové biomarkery biosyntéza   krev   genetika   MeSH
• nádorové buňky kultivované MeSH
• nádorové proteiny biosyntéza   krev   genetika   MeSH
• regulace genové exprese u nádorů imunologie   MeSH
• RNA nádorová genetika   MeSH
• western blotting metody   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• antigeny CD274 MeSH
• CD274 protein, human MeSH Prohlížeč
• messenger RNA MeSH
• nádorové biomarkery MeSH
• nádorové proteiny MeSH
• RNA nádorová MeSH

Specific mutations involving C-terminal part of the nucleolar protein nucleophosmin (NPM) are associated with better outcome of acute myeloid leukemia (AML) therapy, possibly due to aberrant cytoplasmic NPM localization facilitating induction of anti-NPM immune response. Actinomycin D (actD) is known to induce nucleolar stress leading to redistribution of many nucleolar proteins, including NPM. We analyzed the distribution of both wild-type and mutated NPM (NPMmut) in human cell lines, before and after low-dose actD treatment, in living cells expressing exogenous fluorescently labeled proteins as well as using immunofluorescence staining of endogenous proteins in fixed cells. The wild-type NPM form is prevalently nucleolar in intact cells and relocalizes mainly to the nucleoplasm following actD addition. The mutated NPM form is found both in the nucleoli and in the cytoplasm of untreated cells. ActD treatment leads to a marked increase in NPMmut amount in the nucleoplasm while a mild decrease is observed in the cytoplasm. Cell death was induced by low-dose actD in all the studied leukemic cell lines with different p53 and NPM status. In cells expressing the tumor suppresor p53 (CML-T1, OCI-AML3), cell cycle arrest in G1/G0 phase was followed by p53-dependent apoptosis while in p53-null HL60 cells, transient G2/M-phase arrest was followed by cell necrosis. We conclude that although actD does not increase NPM concentration in the cytoplasm, it could improve the effect of standard chemotherapy in leukemias through more general mechanisms.

• Klíčová slova
• ACTINOMYCIN D, APOPTOSIS, LEUKEMIA, MUTATED NUCLEOPHOSMIN, P53-DEFICIENCY,
• MeSH
• apoptóza MeSH
• buněčné jadérko metabolismus   MeSH
• cytoplazma metabolismus   MeSH
• daktinomycin farmakologie   MeSH
• HeLa buňky MeSH
• HL-60 buňky MeSH
• jaderné proteiny genetika   metabolismus   MeSH
• kontrolní body buněčného cyklu účinky léků   MeSH
• leukemie genetika   metabolismus   MeSH
• lidé MeSH
• mutace * MeSH
• nukleofosmin MeSH
• viabilita buněk účinky léků   MeSH
• vztah mezi dávkou a účinkem léčiva MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• daktinomycin MeSH
• jaderné proteiny MeSH
• NPM1 protein, human MeSH Prohlížeč
• nukleofosmin MeSH

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.

• MeSH
• antimetabolity antitumorózní farmakologie   MeSH
• apoptóza účinky léků   MeSH
• azacytidin analogy a deriváty   toxicita   MeSH
• decitabin MeSH
• down regulace účinky léků   MeSH
• HL-60 buňky MeSH
• inhibitory apoptózy metabolismus   MeSH
• kontrolní body fáze G2 buněčného cyklu účinky léků   MeSH
• kontrolní body M fáze buněčného cyklu účinky léků   MeSH
• kyseliny hydroxamové toxicita   MeSH
• lidé MeSH
• lymfocyty cytologie   účinky léků   imunologie   MeSH
• nádorové buněčné linie MeSH
• nádorový supresorový protein p53 nedostatek   genetika   MeSH
• reaktivní formy kyslíku metabolismus   MeSH
• survivin MeSH
• synergismus léků MeSH
• tretinoin farmakologie   MeSH
• vorinostat MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antimetabolity antitumorózní MeSH
• azacytidin MeSH
• BIRC5 protein, human MeSH Prohlížeč
• decitabin MeSH
• inhibitory apoptózy MeSH
• kyseliny hydroxamové MeSH
• nádorový supresorový protein p53 MeSH
• reaktivní formy kyslíku MeSH
• survivin MeSH
• tretinoin MeSH
• vorinostat MeSH

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.

• MeSH
• antimetabolity antitumorózní aplikace a dávkování   MeSH
• apoptóza účinky léků   MeSH
• azacytidin aplikace a dávkování   analogy a deriváty   MeSH
• decitabin MeSH
• kultivované buňky MeSH
• kyseliny hydroxamové aplikace a dávkování   MeSH
• leukemie farmakoterapie   patologie   patofyziologie   MeSH
• lidé MeSH
• lymfocyty cytologie   účinky léků   fyziologie   MeSH
• protokoly antitumorózní kombinované chemoterapie aplikace a dávkování   MeSH
• vorinostat MeSH
• vztah mezi dávkou a účinkem léčiva MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antimetabolity antitumorózní MeSH
• azacytidin MeSH
• decitabin MeSH
• kyseliny hydroxamové MeSH
• vorinostat MeSH