RESUMO
Extracellular signal-regulated kinases (ERK) 1, 2 and 3 are involved in cell proliferation and differentiation, and apoptosis; although ERK1/2 have been widely studied, limited knowledge on ERK3 is available. The present work aimed at investigating ERK3 distribution during cell cycle and apoptosis in human tumor HeLa cells. The analysis performed by double immunofluorescence and immunoelectron microscopy experiments revealed that during interphase ERK3 is mainly resident in the nucleoplasm in association with ribonuclear proteins involved in early pre-mRNA splicing, it undergoes cell cycle-dependent redistribution and, during apoptosis, it remains in the nucleus in the form of massive nuclear aggregates, then moves to the cytoplasm and is finally extruded.
Assuntos
Apoptose/fisiologia , Núcleo Celular/enzimologia , Citoplasma/enzimologia , Interfase/fisiologia , Proteína Quinase 6 Ativada por Mitógeno/metabolismo , Mitose/fisiologia , Células HeLa , Humanos , Transporte Proteico/fisiologiaRESUMO
Poly(ADP-ribose) polymerases are a family of enzymes that catalyze the conversion of NAD+ into ADP-ribose. Among them, Tankyrases have been found to bind to centrosome, mitotic spindle and microsome proteins, in the cytoplasm, and to telomeres in the nucleus, where they play a relevant role in telomere metabolism. However, their precise intracellular localization during interphase has not been so far fully elucidated. We investigated this aspect in situ by double immunofluorescence experiments using antibodies recognizing Tankyrases 1-2 or other proteins residing in specific organelles (Golgi apparatus, mitochondria, lysosomes, endoplasmic reticulum). We used HeLa cells as a model system in vitro, before and after treatment with either actinomycin D or etoposide, to also investigate the possible relocation of Tankyrases during apoptosis. We observed that Tankyrases are distributed both in the nucleus and in the cytoplasm; in this latter compartment, they were found to colocate with the Golgi apparatus but never with the mitochondria; a pool of Tankyrases also colocates with the endoplasmic reticulum and lysosomes. Interestingly, in cells with clear signs of apoptosis, Tankyrases were detectable in the cytoplasmic blebs: this suggests that they are not massively cleaved during apoptosis and persist in the largely heterogeneous apoptotic remnants which are known to contain components of cytoplasmic and nuclear origin.
Assuntos
Interfase/fisiologia , Tanquirases/metabolismo , Antibióticos Antineoplásicos/farmacologia , Antineoplásicos Fitogênicos/farmacologia , Apoptose , Dactinomicina/farmacologia , Retículo Endoplasmático/metabolismo , Etoposídeo/farmacologia , Complexo de Golgi/metabolismo , Células HeLa , Humanos , Transporte Proteico/efeitos dos fármacos , Telomerase/metabolismo , Telômero/metabolismoRESUMO
Cisplatin (cisPt) is a chemotherapeutic drug used for several human malignancies. CisPt cytotoxicity is primarily mediated by its ability to cause DNA damage and subsequent apoptotic cell death. DNA is the primary target of cisPt; however, recent data have shown that cisPt may have important direct interactions with mitochondria, which can induce apoptosis and may account for a significant part of the clinical activity associated with this drug. We have previously demonstrated that in the rat neuronal cell line B50, at 20 h-treatment with cisPt activates apoptosis through an intrinsic pathway involving an alteration of mitochondrial membrane permeability and the release of cytochrome c. The present study investigates different death pathways induced in the same cell line by a prolonged treatment with 40 µM cisPt for 48 h. To address this issue, we focused on caspases-8 and -12, and on the mitochondrial apoptosis inducing factor (AIF), which translocates to the nucleus and induces cell death via caspase-independent pathway. We found that cisPt activates different forms of cell death, i.e. the receptor-mediated apoptotic extrinsic pathway and a death process mediated by endoplasmic reticulum stress. Moreover, we demonstrated that AIF-mediated death occurs, being characterized by the translocation of AIF from mitochondria to the nucleus. On the whole, we provided evidence that prolonged cisPt treatment is able to activate both caspase-dependent and caspase-independent apoptotic pathways in B50 rat neuronal cells.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Cisplatino/farmacologia , Neuroblastoma/tratamento farmacológico , Neuroblastoma/patologia , Animais , Apoptose/fisiologia , Fator de Indução de Apoptose/metabolismo , Caspase 3/metabolismo , Linhagem Celular Tumoral , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , Neuroblastoma/metabolismo , Ratos , Transdução de Sinais/efeitos dos fármacos , Estresse Fisiológico/efeitos dos fármacosRESUMO
OBJECTIVES: Cisplatin (cisPt) is used as a chemotherapeutic agent for the treatment of a variety of human tumours; more recently, it has been demonstrated that tumour cell exposure to cisPt ultimately results in apoptosis, but the mechanism by which nuclear cisPt/DNA generates the cytoplasmic cascade of events involved has not been clarified. We have investigated the effects of cisPt on proliferation in the neuronal cell line B50, with particular attention being given to understand whether mitochondria are a target of cisPt and their involvement in the apoptotic process. MATERIALS AND METHODS: Rat neuronal B50 cells were used to investigate the mechanisms of cisPt-induced cytotoxicity; this line has been used as a model system for neurotoxicity in vivo. RESULTS: Changes in proliferation, induction of apoptosis, activation of caspase-3 and DNA fragmentation were observed in the cells, as well as morphological and biochemical alterations of mithocondria. Activation of caspase-9 confirmed that mitochondria are a target of cisPt. CONCLUSION: CisPt exerts cytotoxic effects in the neuronal B50 cell line via a caspase-dependent pathway with mitochondria being central relay stations.