RESUMO
Human antigen R (HuR) is a RNA-binding protein, which binds to the AU-rich element (ARE) in the 3'-untranslated region (3'-UTR) of certain mRNA and is involved in the export and stabilization of ARE-mRNA. HuR constitutively relocates to the cytoplasm in many cancer cells, however the mechanism of intracellular HuR trafficking is poorly understood. To address this question, we examined the functional role of the cytoskeleton in HuR relocalization. We tested the effect of actin depolymerizing macrolide latrunculin A or myosin II ATPase activity inhibitor blebbistatin for HuR relocalization induced by the vasoactive hormone Angiotensin II in cancer and control normal cells. Western blot and confocal imaging data revealed that both inhibitors attenuated the cytoplasmic HuR in normal cells but no such alteration was observed in cancer cells. Concomitant with changes in intracellular HuR localization, both inhibitors markedly decreased the accumulation and half-lives of HuR target ARE-mRNAs in normal cells, whereas no change was observed in cancer cells. Furthermore, co-immunoprecipitation experiments with HuR proteins revealed clear physical interaction with ß-actin only in normal cells. The current study is the first to verify that cancer cells can implicate a microfilament independent HuR transport. We hypothesized that when cytoskeleton structure is impaired, cancer cells can acquire an alternative HuR trafficking strategy.
Assuntos
Proteína Semelhante a ELAV 1/metabolismo , Neoplasias/metabolismo , Regiões 3' não Traduzidas , Actinas/efeitos dos fármacos , Actinas/metabolismo , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Linhagem Celular Tumoral , Citoplasma/metabolismo , Citoesqueleto/metabolismo , Células HeLa , Células Hep G2 , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Humanos , Miosinas/antagonistas & inibidores , Neoplasias/genética , Ligação Proteica , Transporte Proteico/efeitos dos fármacos , Estabilidade de RNA/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Tiazolidinas/farmacologiaRESUMO
OBJECTIVE: This study evaluated the effect of shortened application time on long-term bond strength with universal adhesives. METHODS AND MATERIALS: Three universal adhesives were used: Clearfil Universal Bond (CU, Kuraray Noritake Dental Inc, Tokyo, Japan), Scotchbond Universal Adhesive (SB, 3M ESPE, St Paul, MN, USA) or G-Premio Bond (GP, GC Corp, Tokyo, Japan). Sixty molars were cut to expose midcoronal dentin and prepared with a regular diamond bur. Each adhesive was applied either according to the manufacturer's instruction or with shortened time. Specimens were stored in distilled water at 37°C for 24 hours and then cut into resin-dentin sticks. Microtensile bond strength (µTBS) was tested after either 24 hours or 1 year of water storage. Data were analyzed by the three-way ANOVA and Duncan tests ( α=0.05). Fracture modes were analyzed under a scanning electron microscope (SEM). One dentin stick per group was selected after fracture mode analysis and further observed using transmission electron microscopy (TEM). Six additional dentin discs were prepared and conditioned with each adhesive under the different application time to observe the adhesive-smear layer interaction by SEM. RESULTS: Shortened application time affected the µTBS ( p<0.001) while storage time did not affect bond strength ( p=0.187). A significant effect of shortened application time on µTBS was observed in the CU at 1 year and in the GP at both storage times. CONCLUSIONS: One-year storage time had no effect on the µTBS of universal adhesives to bur-cut dentin. The performance of universal adhesives can be compromised when applied using a shortened application time.
