RESUMO
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.
Assuntos
Leucemia Mieloide Aguda , Proteína Supressora de Tumor p53 , Humanos , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Apoptose , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Leucemia Mieloide Aguda/metabolismo , Nucléolo Celular/metabolismoRESUMO
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.
RESUMO
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.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Indóis/farmacologia , Leucemia/tratamento farmacológico , Proteínas Nucleares/metabolismo , Linhagem Celular Tumoral , Células HEK293 , Humanos , Leucemia/genética , Leucemia/metabolismo , Proteínas Nucleares/genética , NucleofosminaRESUMO
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 ß1 and αVß3. 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.
Assuntos
Leucemia , Quinases Ativadas por p21 , Adesão Celular , Linhagem Celular , Fibronectinas/genética , Humanos , Leucemia/genética , Quinases Ativadas por p21/genéticaRESUMO
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).
Assuntos
Antígeno B7-H1/sangue , Biomarcadores Tumorais/sangue , Leucemia Mieloide Aguda/sangue , Antígeno B7-H1/genética , Antígeno B7-H1/metabolismo , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Humanos , Leucemia Mieloide Aguda/genética , Mutação , Proteínas Nucleares/genética , Nucleofosmina , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Tirosina Quinase 3 Semelhante a fms/genéticaRESUMO
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.
Assuntos
Nucléolo Celular/metabolismo , Leucemia Mieloide Aguda/metabolismo , Complexos Multiproteicos/metabolismo , Mutação , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Linhagem Celular Tumoral , Nucléolo Celular/genética , Nucléolo Celular/patologia , Células HEK293 , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Complexos Multiproteicos/genética , Proteínas de Neoplasias/genética , Proteínas Nucleares/genética , Nucleofosmina , Fosfoproteínas/genética , Proteínas de Ligação a RNA/genética , NucleolinaRESUMO
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.
Assuntos
Antígeno B7-H1/biossíntese , Biomarcadores Tumorais/biossíntese , Leucemia Mieloide Aguda/imunologia , Processamento Alternativo , Antígeno B7-H1/sangue , Antígeno B7-H1/genética , Biomarcadores Tumorais/sangue , Biomarcadores Tumorais/genética , Western Blotting/métodos , Regulação Neoplásica da Expressão Gênica/imunologia , Variação Genética , Humanos , Leucemia Mieloide Aguda/genética , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/sangue , Proteínas de Neoplasias/genética , RNA Mensageiro/genética , RNA Neoplásico/genética , Células Tumorais CultivadasRESUMO
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.
Assuntos
Dactinomicina/farmacologia , Leucemia/metabolismo , Mutação , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Apoptose , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Nucléolo Celular/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Citoplasma/metabolismo , Relação Dose-Resposta a Droga , Células HL-60 , Células HeLa , Humanos , Leucemia/genética , NucleofosminaRESUMO
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.
Assuntos
Antimetabólitos Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Azacitidina/análogos & derivados , Ácidos Hidroxâmicos/toxicidade , Proteínas Inibidoras de Apoptose/metabolismo , Tretinoína/farmacologia , Proteína Supressora de Tumor p53/genética , Azacitidina/toxicidade , Linhagem Celular Tumoral , Decitabina , Regulação para Baixo/efeitos dos fármacos , Sinergismo Farmacológico , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Células HL-60 , Humanos , Linfócitos/citologia , Linfócitos/efeitos dos fármacos , Linfócitos/imunologia , Pontos de Checagem da Fase M do Ciclo Celular/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Survivina , Proteína Supressora de Tumor p53/deficiência , VorinostatRESUMO
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.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Apoptose/efeitos dos fármacos , Azacitidina/análogos & derivados , Ácidos Hidroxâmicos/administração & dosagem , Leucemia/tratamento farmacológico , Leucemia/fisiopatologia , Linfócitos/efeitos dos fármacos , Antimetabólitos Antineoplásicos/administração & dosagem , Azacitidina/administração & dosagem , Células Cultivadas , Decitabina , Relação Dose-Resposta a Droga , Humanos , Leucemia/patologia , Linfócitos/citologia , Linfócitos/fisiologia , VorinostatRESUMO
Restoration of cellular apoptotic pathways plays a crucial role in cancer therapy strategies. In a broad spectrum of anticancer drugs, epigenetic effectors are in the center of interest mostly because of potential reversibility of their action. Methylation status of the cells is influenced by methyltransferase inhibitor 2-deoxy-5'-azacytidine (decitabine, DAC), but higher concentrations of this agent cause a DNA-damage. In our study, tumor supressor p53-apoptotic pathway was activated in decitabine-induced cell death. Expression of p53-inducible BH3-only apoptotic proteins Puma and Noxa was elevated and large activation of executive caspases was observed. The extent of acetylation in the cell is affected by histonedeacetylase inhibitor suberoylanilide hydroxamic acid (SAHA). Combination of SAHA with decitabine brought synergistic effect on apoptosis triggering in CML-T1 cell line, but apoptosis as well as necrosis occurred also in normal peripheral blood lymphocytes. Therefore, promising potential of such combined therapy calls for more detailed investigation of unwanted effects in normal cells.
Assuntos
Proteínas Reguladoras de Apoptose/genética , Apoptose/efeitos dos fármacos , Azacitidina/análogos & derivados , Ácidos Hidroxâmicos/farmacologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Linfócitos/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas/genética , Antimetabólitos Antineoplásicos/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Azacitidina/farmacologia , Células Cultivadas , Decitabina , Avaliação Pré-Clínica de Medicamentos , Sinergismo Farmacológico , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Linfócitos/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genética , VorinostatRESUMO
Suberoylanilide hydroxamic acid (SAHA) is an inhibitor of histone deacetylases (HDACs) which is being introduced into clinic for the treatment of hematological diseases. We studied the effect of this compound on six human hematopoietic cell lines (JURL-MK1, K562, CML-T1, Karpas-299, HL-60, and ML-2) as well as on normal human lymphocytes and on leukemic primary cells. SAHA induced dose-dependent and cell type-dependent cell death which displayed apoptotic features (caspase-3 activation and apoptotic DNA fragmentation) in most cell types including the normal lymphocytes. At subtoxic concentrations (0.5-1 microM), SAHA increased the cell adhesivity to fibronectin (FN) in all leukemia/lymphoma-derived cell lines but not in normal lymphocytes. This increase was accompanied by an enhanced expression of integrin beta1 and paxillin, an essential constituent of focal adhesion complexes, both at the protein and mRNA level. On the other hand, the inhibition of ROCK protein, an important regulator of cytoskeleton structure, had no consistent effect on SAHA-induced increase in the cell adhesivity. The promotion of cell adhesivity to FN seems to be specific for SAHA as we observed no such effects with other HDAC inhibitors (trichostatin A and sodium butyrate).
Assuntos
Antineoplásicos/farmacologia , Fibronectinas/metabolismo , Ácidos Hidroxâmicos/farmacologia , Leucemia/metabolismo , Linfócitos/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Western Blotting , Adesão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Separação Celular , Citometria de Fluxo , Humanos , Integrina beta1/metabolismo , Linfócitos/metabolismo , Paxilina/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , VorinostatRESUMO
We have examined the effect of suberoylanilide hydroxamic acid (SAHA, Vorinostat, Zolinza) on the viability of normal peripheral blood lymphocytes (PBLs) in vitro and on the expression of 20 apoptosis-related genes. RT-PCR, western blots and flow cytometry were performed to reveal the proteins of apoptosis machinery that were affected to cause cell death. Our data suggest that PBL markedly resisted for approximately 24 h the destructive activity of the agent, but eventually 60% of cells treated with 4 micromol/L SAHA died within 72 h through mitochondrial way of apoptosis. While the expression of the majority of genes remained indifferent against 4 micromol/L SAHA, the cellular levels of BimEL, Bmf-2, Bcl-w and survivin mRNA varied, confirming the pro-apoptotic response of SAHA treated PBL. In addition, the expression of multifunctional proteins c-Myc and p21(WAF1) changed profoundly with the time of SAHA treatment. The Bax activator BimEL increased rapidly, driving cells towards apoptosis likely controlled by c-Myc and p21(WAF1) activities. We suggest that variations in c-Myc and p21(WAF1) expression decelerate the apoptosis in the early period and increase the resistance of resting PBL against SAHA.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Citoproteção , Linfócitos/citologia , Linfócitos/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteína 11 Semelhante a Bcl-2 , Caspases/metabolismo , Morte Celular/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Citocromos c/metabolismo , Citoproteção/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Ácidos Hidroxâmicos/farmacologia , Linfócitos/efeitos dos fármacos , Linfócitos/enzimologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Fosfatidilserinas/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Transporte Proteico/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , VorinostatRESUMO
We have examined the effect of sodium butyrate (SB) on the viability of normal peripheral blood lymphocytes (PBLs) in vitro and the effect of this agent on the expression of 20 apoptosis-related genes. Data suggest that PBL treated with 2 mmol L(-1) SB resisted for at least 8 h the destructive activity of the agent, but eventually 30% of cells died within 72 h. As documented by flow cytometry and cytochrome c release study, cells underwent mitochondrial-derived apoptosis. While the expression of the majority of genes examined by RT-PCR and Western blots remained indifferent to 2 mmol L(-1) SB, the cellular levels of BimEL, c-myc, p53, and p21(WAF1) varied profoundly with the time of SB treatment. The Bax activator BimEL increased rapidly, driving cells toward apoptosis likely controlled by c-myc and p21(WAF1) activities. The c-myc, exercising the role of mediator of the function of BimEL and inhibitor of p21(WAF1) expression, decreased significantly for several hours after adding SB but within 48 h it returned to close to its original value. An apoptosis inhibitor and executive caspase substrate p21(WAF1) increased early at the beginning of treatment but subsequently, within a time frame of 72 h, profoundly dropped in terms of both a caspase-dependent and caspase-independent way. We suggest that variations in c-myc and p21(WAF1) expression delay apoptosis making PBL resistant to SB for several hours, and together with fast catabolism of SB in vivo protect PBL against the destructive activity of this anti-cancerous metabolite of colonic bacteria.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Apoptose/efeitos dos fármacos , Ácido Butírico/farmacologia , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Linfócitos/efeitos dos fármacos , Linfócitos/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Reguladoras de Apoptose/genética , Proteína 11 Semelhante a Bcl-2 , Caspases/metabolismo , Células Cultivadas , Colágeno Tipo XI/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/genética , Citocromos c/metabolismo , Ativação Enzimática , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Membranas Intracelulares/efeitos dos fármacos , Proteínas de Membrana/genética , Mitocôndrias/efeitos dos fármacos , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-myc/genética , Espécies Reativas de Oxigênio/metabolismoRESUMO
The present study was undertaken to provide information on the nucleolar and cytoplasmic density in specimens stained for RNA during "cell dedifferentiation" represented by blastic transformation of mature T lymphocytes. Nucleolar and cytoplasmic RNA's were visualized using a simple cytochemical method followed by computer assisted densitometry and size measurements of digitised images. An increased nucleolar and cytoplasmic RNA density accompanying the blastic transformation was significant after 48 hours of cultivation with phytohemaglutinin (PHA) when stimulated cells were characterized the largest nucleolar size reflecting S or G2 phase of the cell cycle. On the other hand, significantly larger ratio of the nucleolar to cytoplasmic density was noted only after a shorter cultivation when stimulated cells were presumably in the G1 phase. Thus the increased nucleolar and cytoplasmic RNA density together represented an accompanying phenomenon of the cell proliferation and cycling state. From the methodical point of view, the nucleolar and cytoplasmic RNA densitometry appeared as a simple as well as useful additional method to study "dedifferentiation" or various cell states at the single cell level. In addition, it was also interesting that the increase of the nucleolar diameter in stimulated cells was much larger than that of the nucleolar density. Such difference suggested that the RNA content in nucleoli was related mainly to their size.
Assuntos
Desdiferenciação Celular , Nucléolo Celular/metabolismo , Citoplasma/metabolismo , Ativação Linfocitária/imunologia , RNA/metabolismo , Linfócitos T/citologia , Linfócitos T/imunologia , Humanos , Imuno-Histoquímica , Tamanho das OrganelasRESUMO
We have examined the ability of actinomycin D to induce apoptosis in human peripheral blood lymphocytes. Run-On assays were performed to specify the primary molecular damage, reverse transcription-PCR, Western blots and flow cytometry studies were performed to ascertain which proteins of the apoptosis machinery were affected to cause actinomycin D-induced cell death. Expression of 23 apoptosis-related genes was investigated. The down-regulation of ribosomal RNA synthesis caused by actinomycin D induced a mitochondria-dependent apoptosis. Although the expression of the majority of examined genes remained indifferent against actinomycin D activity, the cellular level of p53 protein increased, subsequently upregulating both Puma mRNA and protein. Puma-mediated mitochondrial apoptosis was accompanied by nucleolin cleavage and Bcl-2 mRNA destabilization. The stability of the cellular level of Bcl-2 protein independent of a mRNA decrease suggests that protection of Bcl-2 protein against proteasomal degradation can moderate the apoptotic process. In peripheral blood lymphocytes cultured in vitro, the apoptosis induced by a low concentration of actinomycin D (10 nmol/l) is dependent on p53 and Puma activation. This apoptotic pathway is demonstrated in peripheral blood lymphocytes for the first time. A different apoptotic pathway induced in peripheral blood lymphocytes using this drug has, however, been previously revealed by other authors. The combination of cell specificity and dose-dependent effects can likely play a decisive role in apoptosis observed in peripheral blood lymphocytes after genotoxic drug application.