RESUMO
Pollution generated by deposition of industrial activity waste in the environment without due care can lead to serious environmental consequences. Bioassays in higher plants are means of understanding the cytogenotoxic effects of these substances. In the present work, Allium cepa L. was used as a model species to assess nucleolar changes induced by environmental pollutants. The substances used were Methyl Methane Sulfonate (MMS), cadmium (Cd), Spent Potliner (SPL) and the herbicide Atrazine. Water was used as a negative control. The silver-stained nucleolar organizer region (AgNOR) assay was used making it possible to evaluate how nucleolar parameters (number of nucleoli per nucleus and nucleoli area) behave when facing stress caused by such pollutants. The results obtained showed a variation in the observed parameters: an increase in the number of nucleoli in the treated cells and tendency to a reduction in nucleolar area, indicating that the tested pollutants may have impaired nucleolar activity. In addition, it was possible to establish a relationship between the behavior of the nucleolus with other changes as plantlet growth, cell proliferation, and DNA damage.
Assuntos
Nucléolo Celular/efeitos dos fármacos , Monitoramento Ambiental/métodos , Poluentes Ambientais/toxicidade , Resíduos Industriais/efeitos adversos , Mutagênicos/toxicidade , Nucléolo Celular/patologia , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/patologia , Biomarcadores Ambientais/efeitos dos fármacos , Cebolas/citologia , Cebolas/efeitos dos fármacosRESUMO
Nucleolar alterations resulting from the action of either chemical or physical agents can serve as important genotoxicity biomarkers. In this study, the efficiency of AgNOR banding technique to identify the presence of nucleoli in micronucleus and assess nucleolar alterations in aberrant cells of Allium cepa was evaluated. Seeds of this plant were exposed to both water samples from a river that receives untreated urban effluent and to the trifluralin herbicide (0.84 mg/L concentration), both analyzed in two different seasons (summer and winter seasons). Samples induced significant frequencies of chromosomal and nuclear aberrations and micronuclei, as observed in cells submitted to conventional chromosomal staining. The herbicide caused a significant increase in the number of nucleoli and micronuclei, interpreted as due to the elimination of excessive nucleolar material resulting from polyploidization. The use of the AgNOR technique enabled the identification of both the presence of the nucleolus in some micronuclei and the nucleolar organizer region (NOR) behavior of aberrant cells. The NOR-banding technique showed to be an efficient tool for studying the genotoxic effects caused by a xenobiotics and a complex environmental sample.
Assuntos
Nucléolo Celular/efeitos dos fármacos , Herbicidas/toxicidade , Cebolas/efeitos dos fármacos , Trifluralina/toxicidade , Poluentes Químicos da Água/toxicidade , Brasil , Cromossomos de Plantas/genética , Cidades , Dano ao DNA , Herbicidas/análise , Cebolas/citologia , Cebolas/genética , Rios/química , Trifluralina/análise , Águas Residuárias/química , Poluentes Químicos da Água/análiseRESUMO
It has been established that retinoids exert some of their effects on cell differentiation and malignant phenotype reversion through the interaction with different members of the protein kinase C (PKC) family. Till nowadays the nature and extension of this interaction is not well understood. Due to the cytostatic and differentiating effects of retinoids, in the present study we propose to evaluate whether the crosstalk between the retinoid system and the PKC pathway could become a possible target for breast cancer treatment. We could determine that ATRA (all-trans retinoic) treatment showed a significant growth inhibition due to (G1 or G2) cell cycle arrest both in LM3 and SKBR3, a murine and human mammary cell line respectively. ATRA also induced a remarkable increase in PKCα and PKCδ expression and activity. Interestingly, the pharmacological inhibition of these two PKC isoforms prevented the activation of retinoic acid receptors (RARs) by ATRA, indicating that both PKC isoforms are required for RARs activation. Moreover, PKCδ inhibition also impaired ATRA-induced RARα translocation to the nucleus. In vivo assays revealed that a combined treatment using ATRA and PKCα inhibitors prevented lung metastatic dissemination in an additive way. Our results clearly indicate that ATRA modulates the expression and activity of different PKCs. Besides inducing cell arrest, the activity of both PKC is necessary for the induction of the retinoic acid system. The combined ATRA and PKCα inhibitors could be an option for the hormone-independent breast cancer treatment.
Assuntos
Neoplasias da Mama/metabolismo , Proteína Quinase C-alfa/metabolismo , Proteína Quinase C-delta/metabolismo , Receptores do Ácido Retinoico/metabolismo , Tretinoína/metabolismo , Animais , Neoplasias da Mama/tratamento farmacológico , Diferenciação Celular/efeitos dos fármacos , Nucléolo Celular/efeitos dos fármacos , Nucléolo Celular/metabolismo , Feminino , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/metabolismo , Proteína Quinase C-alfa/antagonistas & inibidores , Proteína Quinase C-delta/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Células Tumorais CultivadasRESUMO
CIGB-300 is a novel anticancer peptide that impairs the casein kinase 2-mediated phosphorylation by direct binding to the conserved phosphoacceptor site on their substrates. Previous findings indicated that CIGB-300 inhibits tumor cell proliferation in vitro and induces tumor growth delay in vivo in cancer animal models. Interestingly, we had previously demonstrated that the putative oncogene B23/nucleophosmin (NPM) is the major intracellular target for CIGB-300 in a sensitive human lung cancer cell line. However, the ability of this peptide to target B23/NPM in cancer cells with differential CIGB-300 response phenotype remained to be determined. Interestingly, in this work, we evidenced that CIGB-300's antiproliferative activity on tumor cells strongly correlates with its nucleolar localization, the main subcellular localization of the previously identified B23/NPM target. Likewise, using CIGB-300 equipotent doses (concentration that inhibits 50% of proliferation), we demonstrated that this peptide interacts and inhibits B23/NPM phosphorylation in different cancer cell lines as evidenced by in vivo pull-down and metabolic labeling experiments. Moreover, such inhibition was followed by a fast apoptosis on CIGB-300-treated cells and also an impairment of cell cycle progression mainly after 5 h of treatment. Altogether, our data not only validates B23/NPM as a main target for CIGB-300 in cancer cells but also provides the first experimental clues to explain their differential antiproliferative response. Importantly, our findings suggest that further improvements to this cell penetrating peptide-based drug should entail its more efficient intracellular delivery at such subcellular localization.
Assuntos
Antineoplásicos/farmacologia , Nucléolo Celular/efeitos dos fármacos , Peptídeos Cíclicos/farmacologia , Antineoplásicos/metabolismo , Apoptose , Caseína Quinase II/antagonistas & inibidores , Pontos de Checagem do Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Concentração Inibidora 50 , Proteínas Nucleares/metabolismo , Nucleofosmina , Peptídeos Cíclicos/metabolismo , Fosforilação , Processamento de Proteína Pós-Traducional/efeitos dos fármacosRESUMO
Crotamine, a low molecular weight cationic polypeptide from the venom of the South American rattlesnake Crotalus durissus terrificus is a natural cell-penetrating peptide with functional versatility. The presence of nine lysine residues and three disulfide bonds renders crotamine highly compact, stable and positively charged. Topologically, crotamine adopts an ancient ß-defensin fold that is found in diverse families of endogenous and venom polypeptides dedicated to host defense. Crotamine is unique among several classes of bioactive peptides because it possesses both cell penetrating and antimicrobial activities and selective biological action toward some cell types at a given cell cycle phase. Because it can rapidly and efficiently translocate into actively proliferating cells, crotamine is being investigated for labeling highly replicating cells and for use as a chemotherapeutic adjuvant. Peptides derived from crotamine, nucleolar targeting peptides (NrTPs), have been designed and are being studied. NrTPs retain some crotamine properties, such as efficient cellular uptake and preferential nuclear localization whereas they improve upon other properties. For example, NrTPs are smaller than crotamine, show higher preferential nucleolar localization, and better facilitate ZIP-code localization of therapeutic proteins.
Assuntos
Peptídeos Catiônicos Antimicrobianos/isolamento & purificação , Antineoplásicos/isolamento & purificação , Peptídeos Penetradores de Células/isolamento & purificação , Venenos de Crotalídeos/química , Venenos de Crotalídeos/isolamento & purificação , Proteínas de Répteis/química , Sequência de Aminoácidos , Animais , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/farmacologia , Antineoplásicos/química , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Nucléolo Celular/efeitos dos fármacos , Nucléolo Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Peptídeos Penetradores de Células/química , Peptídeos Penetradores de Células/farmacologia , Venenos de Crotalídeos/farmacologia , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Secundária de ProteínaRESUMO
NIP7 is one of the many trans-acting factors required for eukaryotic ribosome biogenesis, which interacts with nascent pre-ribosomal particles and dissociates as they complete maturation and are exported to the cytoplasm. By using conditional knockdown, we have shown previously that yeast Nip7p is required primarily for 60S subunit synthesis while human NIP7 is involved in the biogenesis of 40S subunit. This raised the possibility that human NIP7 interacts with a different set of proteins as compared to the yeast protein. By using the yeast two-hybrid system we identified FTSJ3, a putative ortholog of yeast Spb1p, as a human NIP7-interacting protein. A functional association between NIP7 and FTSJ3 is further supported by colocalization and coimmunoprecipitation analyses. Conditional knockdown revealed that depletion of FTSJ3 affects cell proliferation and causes pre-rRNA processing defects. The major pre-rRNA processing defect involves accumulation of the 34S pre-rRNA encompassing from site A' to site 2b. Accumulation of this pre-rRNA indicates that processing of sites A0, 1 and 2 are slower in cells depleted of FTSJ3 and implicates FTSJ3 in the pathway leading to 18S rRNA maturation as observed previously for NIP7. The results presented in this work indicate a close functional interaction between NIP7 and FTSJ3 during pre-rRNA processing and show that FTSJ3 participates in ribosome synthesis in human cells.
Assuntos
Metiltransferases/metabolismo , Proteínas Nucleares/metabolismo , Precursores de RNA/metabolismo , Processamento Pós-Transcricional do RNA , Nucléolo Celular/efeitos dos fármacos , Nucléolo Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Doxiciclina/farmacologia , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Imunoprecipitação , Cinética , Metiltransferases/genética , Metiltransferases/isolamento & purificação , Proteínas Nucleares/genética , Proteínas Nucleares/isolamento & purificação , Ligação Proteica/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , RNA/metabolismo , Interferência de RNA/efeitos dos fármacos , Processamento Pós-Transcricional do RNA/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reprodutibilidade dos TestesRESUMO
In this work we show that under Actinomycin D (ActD) treatment, several RNA Binding Proteins (RBPs) involved in mRNA metabolism are relocalized into the nucleolus in Trypanosoma cruzi as a specific stress response. ATP depletion as well as kinase inhibition markedly reduced the nucleolar localization response, suggesting that an energy-dependent transport modulated by the phosphorylation status of the parasite might be required. Deletion analyses in one of such proteins, TcSR62, showed that a domain bearing basic amino acids located in the COOH terminal region was sufficient to promote its nucleolar relocalization. Interestingly, we showed that in addition to RBPs, poly(A)+ RNA is also accumulated into the nucleolus in response to ActD treatment. Finally, we found out that nucleolar relocalization of RBPs is also triggered by severe heat shock in a reversible way. Together, these results suggest that the nucleolus of an early divergent eukaryote is either able to sequester key factors related to mRNA metabolism in response to transcriptional stress or behaves as a RBP processing center, arguing in favour to the hypothesis that the non-traditional features of the nucleolus could be acquired early during evolution.
Assuntos
Nucléolo Celular/efeitos dos fármacos , Nucléolo Celular/metabolismo , Dactinomicina/farmacologia , Proteínas de Ligação a RNA/metabolismo , Trypanosoma cruzi/efeitos dos fármacos , Trypanosoma cruzi/metabolismo , Transporte Proteico/efeitos dos fármacosRESUMO
Our group is interested in rRNA and ribosome biogenesis in the parasitic protozoan Trypanosoma cruzi. Epimastigotes represent an extracellular replicative stage of T. cruzi and can be cultured in axenic media. The growth curve of epimastigotes allows assessment of potential differences in the nucleoli of cells undergoing growth-rate transitions. To establish cellular parameters for studying ribosome biogenesis in T. cruzi, a morphometric analysis of the nucleoli of cultured cells in the exponential and stationary phases was conducted. Electron micrograph-based measurements of nuclear sections from independent cells demonstrated that the nucleolar area is over twofold higher in exponentially growing cells, as compared with epimastigotes in the stationary phase. The granular component of the nucleoli of actively growing cells was the main structural element. Cycloheximide moderately reduced the apparent size of the nucleoli without an apparent disruption of their architecture. Our results provide a firm basis for the establishment of an experimental model to study the organization of the nucleolus during the growth and development of T. cruzi.
Assuntos
Trypanosoma cruzi/citologia , Nucléolo Celular/efeitos dos fármacos , Nucléolo Celular/ultraestrutura , Cicloeximida/farmacologia , Inibidores da Síntese de Proteínas/farmacologia , Trypanosoma cruzi/efeitos dos fármacos , Trypanosoma cruzi/ultraestruturaRESUMO
Oestrogen exposure during the early post-natal period affects male growth, physiology, and susceptibility to disease in adult life. The prostate gland is susceptible to this oestrogen imprinting, showing a reduced expression of the androgen receptor and inability to respond to androgen stimulus. In this context, we decided to study key signalling regulators of ventral prostate (VP) functioning after early postnatal exposure to high-dose oestrogen. Our results showed a decrease of mTOR phosphorylation and its direct downstream target 4EBP. It is known that mTOR-induced signalling is a pivotal pathway of cell metabolism, which is able to control gene transcription and protein synthesis. We then decided to investigate other indicators of a reduced metabolism in the oestrogenized prostate, and found that the luminal epithelial cells were shorter, less polarized and had smaller nuclei containing more compacted chromatin, suggesting that a general mechanism of regulating gene expression and protein synthesis could be installed in the epithelium of the oestrogenized VP. To evaluate this idea, we analysed nucleolar morphology, and measured the amount of ribosomes and the level of methylation of the 45S ribosomal RNA promoter region. These data indicated that the nucleolus was dismantled and that the methylation at the 45S promoter was increased ( approximately five-fold). Taken together, the results support the idea that the oestrogenized prostate maintains a very low transcriptional level and protein turnover by affecting canonical signalling pathways and promoting nuclear and nucleolar changes.
Assuntos
Estrogênios/fisiologia , Impressão Genômica , Próstata/metabolismo , Biossíntese de Proteínas/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacos , Animais , Nucléolo Celular/efeitos dos fármacos , DNA Ribossômico/metabolismo , Células Epiteliais/citologia , Masculino , Próstata/efeitos dos fármacos , Ratos , Ratos Wistar , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/efeitos dos fármacosRESUMO
(1) Following acute spinal cord injury, progesterone modulates several molecules essential for motoneuron function, although the morphological substrates for these effects are unknown. (2) The present study analyzed morphological changes in motoneurons distal to the lesion site from rats with or without progesterone treatment. We employed electron microscopy to study changes in nucleus and cytoplasm and immunohistochemistry for the microtubule-associated protein 2 (MAP2) for changes in cytoskeleton. (3) After spinal cord injury, the nucleoplasm appeared more finely dispersed resulting in reduced electron opacity and the nucleus adopted an eccentric position. Changes of perikarya included dissolution of Nissl bodies and dissociation of polyribosomes (chromatolysis). After progesterone treatment for 3 days, the deafferented motoneurons now presented a clumped nucleoplasm, a better-preserved rough endoplasmic reticulum and absence of chromatolysis. Progesterone partially prevented development of nuclear eccentricity. Whereas 50% of injured motoneurons showed nuclear eccentricity, only 16% presented this phenotype after receiving progesterone. Additionally, injured rats showed reduced immunostaining for MAP2 in dendrites, pointing to cytoskeleton abnormalities, whereas progesterone treatment attenuated the injury-induced loss of MAP2. (4) Our data indicated that progesterone maintained in part neuronal ultrastructure, attenuated chromatolysis, and preclude the loss of MAP2, suggesting a protective effect during the early phases of spinal cord injury.