RESUMEN
We have previously shown that suberoylanilide hydroxamic acid (SAHA) treatment increases the adhesivity of leukemic cells to fibronectin at clinically relevant concentrations. Now, we present the results of the proteomic analysis of SAHA effects on leukemic cell lines using 2-DE and ProteomLab PF2D system. Histone acetylation at all studied acetylation sites reached the maximal level after 5 to 10 h of SAHA treatment. No difference in histone acetylation between subtoxic and toxic SAHA doses was observed. SAHA treatment induced cofilin phosphorylation at Ser3, an increase in vimentin and paxillin expression and a decrease in stathmin expression as confirmed by western-blotting and immunofluorescence microscopy. The interaction of cofilin with 14-3-3 epsilon was documented using both Duolink system and coimmunoprecipitation. However, this interaction was independent of cofilin Ser3 phosphorylation and the amount of 14-3-3-ε-bound cofilin did not rise following SAHA treatment. SAHA-induced increase in the cell adhesivity was associated with an increase in PAK phosphorylation in CML-T1 cells and was abrogated by simultaneous treatment with IPA-3, a PAK inhibitor. The effects of SAHA on JURL-MK1 cells were similar to those of other histone deacetylase inhibitors, tubastatin A and sodium butyrate. The proteome analysis also revealed several potential non-histone targets of histone deacetylases.
Asunto(s)
Antineoplásicos/farmacología , Ácidos Hidroxámicos/farmacología , Leucemia/metabolismo , Proteínas de Neoplasias/metabolismo , Proteoma/metabolismo , Acetilación/efectos de los fármacos , Adhesión Celular/efectos de los fármacos , Células HL-60 , Humanos , Células K562 , Leucemia/tratamiento farmacológico , Leucemia/patología , Fosforilación/efectos de los fármacos , Factores de Tiempo , VorinostatRESUMEN
Neutrophil gelatinase associated lipocalin belongs to a family of small proteins, lipocalins, engaged in the transmembrane transportation of lipophylic substances. Originally isolated from specific granules of neutrophils, it was later located in bone marrow cells as well as lung, bronchial and colon epithelial cells. The expression of neutrophil lipocalin in epithelial cells and in body fluids considerably augments during the occurrence of inflammations and some cancers. A modulation of immunity response was thus suggested to be the main function of neutrophil lipocalin as well as the bacteriostatic effect originating from competition between neutrophil lipocalin and bacteria for siderophoric iron. Forming protective complexes with gelatinase B, the neutrophil lipocalin is implicated in regulatory processes of physiological and pathological rebuilding of tissues, mainly in the angiogenesis. The determination of neutrophil lipocalin levels in body fluids able to discriminate between bacterial and viral infections provides a powerful diagnostic tool. The examination of neutrophil lipocalin in the sera and urine of patients at risk of renal failure offers a very early marker of this acute state. Neutrophil lipocalin represents a sensitive non-invasive marker of renal ischemia and in patients with cystic fibrosis the marker of acute pulmonary exacerbation. Discussions have been conducted regarding the role of neutrophil lipocalin as an early marker of pancreatic cancer or of neutrophilic activation in severe cases of bowel diseases.
Asunto(s)
Proteínas de Fase Aguda , Proteínas Proto-Oncogénicas , Proteínas de Fase Aguda/análisis , Proteínas de Fase Aguda/química , Proteínas de Fase Aguda/fisiología , Proteínas de Fase Aguda/uso terapéutico , Biomarcadores/análisis , Humanos , Lipocalina 2 , Lipocalinas , Proteínas Proto-Oncogénicas/análisis , Proteínas Proto-Oncogénicas/química , Proteínas Proto-Oncogénicas/fisiología , Proteínas Proto-Oncogénicas/uso terapéuticoRESUMEN
BACKGROUND: Natural lectin, phytohemagglutinin, initiates the transformation of normally quiescent T lymphocytes into proliferating lymphoblast-like cells. Recently we have shown that the transformation is accompanied by strong promotion of ribosomal RNA synthesis and by phosphorylation of its activator, initiating factor UBF, both culminating in a synthetic phase of the first cell division cycle. In contrast we have revealed that the UBF gene was activated and its transcription culminated in the early G1 phase. We examined three possible delaying mechanisms: the kinetics of unwinding of rDNA chromatin, the kinetics of transcription of genes coding for the second initiating factor, SL1 complex, and the kinetics of the translation of UBF protein product. METHODS AND RESULTS: Up to 48 hrs following the addition of phytohemagglutinin to the growth medium, we monitored structural changes in the rDNA chromatin using indirect antiUBF immunofluorescence. The data indicated an increased number of separated transcriptional units during the G1 phase of the first cycle. In a time interval of up to 70 hrs we measured the mRNA levels of four constituents of SL1 complex: TAF110, TAF63, TAF48 and TBP using the RT-PCR method. We found a close correlation between the kinetics of the transcription of UBF and SL1 genes and the maximal rate in the early G1 phase. Using metabolic labelling with 35S methionine/cysteine we monitored the translation of UBF protein in PHA stimulated lymphocytes. The data suggested that UBF translation, starting in the S phase, paralleled chromosomal DNA replication. CONCLUSIONS: During the transformation of normal T lymphocytes into proliferating blast-like cells, the multicopy rDNA gene unwinds in the G1 phase of the first cycle forming individual transcriptional units. Genes coding for factors which initiate synthesis of ribosomal precursors are activated in the early G1 phase. The G1 synthesis of ribosomal RNA is accelerated by phosphorylation of the hypophosphorylated UBF pool. As blastic transformation develops UBF translation is triggered in the S phase and neosynthesized UBF, activated by phosphorylation, pushes the synthesis of ribosomal precursors to maximal efficiency. The process of blastic transformation interferes throughout the entire prolonged G1 phase of the first cell division cycle.