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1.
Cancers (Basel) ; 12(8)2020 Aug 04.
Artigo em Inglês | MEDLINE | ID: mdl-32759730

RESUMO

Induction of mitotic catastrophe through the disruption of microtubules is an established target in cancer therapy. However, the molecular mechanisms determining the mitotic catastrophe and the following apoptotic or non-apoptotic cell death remain poorly understood. Moreover, many existing drugs targeting tubulin, such as vincristine, have reduced efficacy, resulting from poor solubility in physiological conditions. Here, we introduce a novel small molecule 2-aminoimidazoline derivative-OAT-449, a synthetic water-soluble tubulin inhibitor. OAT-449 in a concentration range from 6 to 30 nM causes cell death of eight different cancer cell lines in vitro, and significantly inhibits tumor development in such xenograft models as HT-29 (colorectal adenocarcinoma) and SK-N-MC (neuroepithelioma) in vivo. Mechanistic studies showed that OAT-449, like vincristine, inhibited tubulin polymerization and induced profound multi-nucleation and mitotic catastrophe in cancer cells. HeLa and HT-29 cells within 24 h of treatment arrested in G2/M cell cycle phase, presenting mitotic catastrophe features, and 24 h later died by non-apoptotic cell death. In HT-29 cells, both agents altered phosphorylation status of Cdk1 and of spindle assembly checkpoint proteins NuMa and Aurora B, while G2/M arrest and apoptosis blocking was consistent with p53-independent accumulation in the nucleus and largely in the cytoplasm of p21/waf1/cip1, a key determinant of cell fate programs. This is the first common mechanism for the two microtubule-dissociating agents, vincristine and OAT-449, determining the cell death pathway following mitotic catastrophe demonstrated in HT-29 cells.

2.
J Vis Exp ; (159)2020 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-32478714

RESUMO

Transgenic animal models are fundamentally important for modern biomedical research. The incorporation of foreign genes into early mouse or rat embryos is an invaluable tool for gene function analysis in living organisms. The standard transgenesis method is based on microinjecting foreign DNA fragments into a pronucleus of a fertilized oocyte. This technique is widely used in mice but remains relatively inefficient and technically demanding in other animal species. The transgene can also be introduced into one-cell-stage embryos via lentiviral infection, providing an effective alternative to standard pronuclear injections, especially in species or strains with a more challenging embryo structure. In this approach, a suspension that contains lentiviral vectors is injected into the perivitelline space of a fertilized rat embryo, which is technically less demanding and has a higher success rate. Lentiviral vectors were shown to efficiently incorporate the transgene into the genome to determine the generation of stable transgenic lines. Despite some limitations (e.g., Biosafety Level 2 requirements, DNA fragment size limits), lentiviral transgenesis is a rapid and efficient transgenesis method. Additionally, using female rats that are mated with a fertile male strain with a different dominant fur color is presented as an alternative to generate pseudopregnant foster mothers.


Assuntos
Vetores Genéticos/genética , Lentivirus/genética , Animais , Camundongos , Ratos , Ratos Transgênicos
3.
Neurobiol Dis ; 130: 104499, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31176717

RESUMO

TAR DNA-binding protein 43 (TDP-43) is a hallmark of some neurodegenerative disorders, such as frontotemporal lobar degeneration and amyotrophic lateral sclerosis. TDP-43-related pathology is characterized by its abnormally phosphorylated and ubiquitinated aggregates. It is involved in many aspects of RNA processing, including mRNA splicing, transport, and translation. However, its exact physiological function and role in mechanisms that lead to neuronal degeneration remain elusive. Transgenic rats that were characterized by TDP-43 depletion in neurons exhibited enhancement of the acquisition of fear memory. At the cellular level, TDP-43-depleted neurons exhibited a decrease in the short-term plasticity of intrinsic neuronal excitability. The induction of long-term potentiation in the CA3-CA1 areas of the hippocampus resulted in more stable synaptic enhancement. At the molecular level, the protein levels of an unedited (R) FLOP variant of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) GluR1 and GluR2/3 subunits decreased in the hippocampus. Alterations of FLOP/FLIP subunit composition affected AMPAR kinetics, reflected by cyclothiazide-dependent slowing of the decay time of AMPAR-mediated miniature excitatory postsynaptic currents. These findings suggest that TDP-43 may regulate activity-dependent neuronal plasticity, possibly by regulating the splicing of genes that are responsible for fast synaptic transmission and membrane potential.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Hipocampo/metabolismo , Memória/fisiologia , Plasticidade Neuronal/fisiologia , Neurônios/metabolismo , Animais , Proteínas de Ligação a DNA/genética , Espinhas Dendríticas/metabolismo , Ratos , Ratos Transgênicos , Receptores de AMPA/metabolismo , Transmissão Sináptica/fisiologia
4.
Oncotarget ; 8(6): 9303-9322, 2017 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-28030837

RESUMO

Anticancer therapies that induce DNA damage tend to trigger senescence in cancer cells, a process known as therapy-induced senescence (TIS). Such cells may undergo atypical divisions, thus contributing to tumor re-growth. Accumulation of senescent cancer cells reduces survival of patients after chemotherapy. As senescence interplays with autophagy, a dynamic recycling process, we sought to study whether inhibition of autophagy interferes with divisions of TIS cells. We exposed human colon cancer HCT116 cells to repeated cycles of a chemotherapeutic agent - doxorubicin (doxo) and demonstrated induction of hallmarks of TIS (e.g. growth arrest, hypertrophy, poliploidization and secretory phenotype) and certain properties of cancer stem cells (increased NANOG expression, percentages of CD24+ cells and side population). Colonies of small and highly proliferative progeny appeared shortly after drug removal. Treatment with bafilomycin A1 (BAF A1), an autophagy inhibitor, postponed short term in vitro cell re-population. It was associated with reduction in the number of diploid and increase in the number of poliploid cells. In a long term, a pulse of BAF A1 resulted in reactivation of autophagy in a subpopulation of HCT116 cells and increased proliferation. Accordingly, the senescent HCT116 cells treated with BAF A1 when injected into NOD/SCID mice formed tumors, in contrast to the controls.Our results suggest that senescent cancer cells that appear during therapy, can be considered as dormant cells that contribute to cancer re-growth, when chemotherapeutic treatment is stopped. These data unveil new mechanisms of TIS-related cancer maintenance and re-population, triggered by a single pulse of BAF A1 treatment.


Assuntos
Antibióticos Antineoplásicos/farmacologia , Proliferação de Células/efeitos dos fármacos , Senescência Celular/efeitos dos fármacos , Neoplasias do Colo/tratamento farmacológico , Doxorrubicina/farmacologia , Macrolídeos/farmacologia , Células-Tronco Neoplásicas/efeitos dos fármacos , Células da Side Population/efeitos dos fármacos , Animais , Autofagia/efeitos dos fármacos , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Feminino , Regulação Neoplásica da Expressão Gênica , Células HCT116 , Humanos , Camundongos Endogâmicos NOD , Camundongos SCID , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Fenótipo , Ploidias , Células da Side Population/metabolismo , Células da Side Population/patologia , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , Carga Tumoral
5.
Sci Rep ; 6: 28209, 2016 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-27312902

RESUMO

Whole-brain imaging with light-sheet fluorescence microscopy and optically cleared tissue is a new, rapidly developing research field. Whereas successful attempts to clear and image mouse brain have been reported, a similar result for rats has proven difficult to achieve. Herein, we report on creating novel transgenic rat harboring fluorescent reporter GFP under control of neuronal gene promoter. We then present data on clearing the rat brain, showing that FluoClearBABB was found superior over passive CLARITY and CUBIC methods. Finally, we demonstrate efficient imaging of the rat brain using light-sheet fluorescence microscopy.


Assuntos
Mapeamento Encefálico/métodos , Encéfalo/diagnóstico por imagem , Imageamento Tridimensional/métodos , Microscopia de Fluorescência/métodos , Animais , Proteínas de Fluorescência Verde/genética , Neurônios/citologia , Regiões Promotoras Genéticas/genética , Ratos , Ratos Transgênicos , Ratos Wistar , Antígenos Thy-1/genética , Antígenos Thy-1/metabolismo
6.
Postepy Biochem ; 61(2): 159-67, 2015.
Artigo em Polonês | MEDLINE | ID: mdl-26689008

RESUMO

TDP-43 is mainly a nuclear protein belonging to the heterogeneous ribonucleoproteins family. It plays a crucial role in the regulation of gene transcription and RNA processing. In 2006, TDP-43 was characterized as the main component of ubiquitin-positive inclusions observed in Frontotemporal Lobar Degeneration (FTLD) and Amyotrophic Lateral Sclerosis (ALS) cases. Since then, its central role in a number of neurodegenerative diseases was confirmed, originating the TDP-43 proteinopathies term. Pathological TDP-43 redistributes and accumulates in the cytoplasm forming toxic aggregates. Plethora of animal models recapitulating features typical for human TDP-43 proteinopathies has been generated. However, the mechanism involving TDP-43 and causing functional disturbances, like dementia and motoneurons degeneration, remains unknown. Loss and gain of function hypotheses are proposed, but they still need to be verified.


Assuntos
Esclerose Lateral Amiotrófica/fisiopatologia , Proteínas de Ligação a DNA/fisiologia , Degeneração Lobar Frontotemporal/fisiopatologia , Neurônios/patologia , Processamento Pós-Transcricional do RNA/fisiologia , Transcrição Gênica/fisiologia , Humanos
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