RESUMEN
We cultured silver pomfret for 20 days, decreasing water temperature from 18 to 8 â, and sampled muscle every 5 days. Muscle fiber degeneration and apoptosis began to increase at 13 â detected by HE and TUNEL staining. Further analysis of transcriptome revealed that several apoptosis-related pathways were highly enriched by differentially expressed genes (DEGs). We analyzed 10 DEGs from these pathways by RT-qPCR during the temperature-decreasing process. JNK1, PIDD, CytC, Casp 3, and GADD45 were up-regulated after 15 and 20 days, while DUSP3, JNK2, and PARP genes were down-regulated after 15 and 20 days. DUSP5 was up-regulated from 10 to 20 days, and C-JUN was up-regulated after 20 days. We analyzed apoptosis in PaM cells under different temperatures (26 â, 23 â, 20 â, 17 â, and 14 â). The cell viability significantly declined from 14 to 20 â; the TUNEL and IHC results showed that the apoptosis signal increased with the temperature dropping, especially in 17 â and 14 â; DUSP5, JNK1, CytC, C-JUN, Casp 3, and GADD45 were up-regulated at 17 â and 14 â, and PIDD was up-regulated at 20 â, 17 â, and 14 â. DUSP3 was up-regulated at 20 â but down-regulated at 17 â and 14 â, and PARP was down-regulated at 17 â and 14 â. JNK2 was up-regulated at 20 â but down-regulated at 17 â and 14 â. Our results suggest that DUSP could help inhibit apoptosis in the initial stage of cold stress, but low temperature could down-regulate it and up-regulate JNK-C-JUN, inducing apoptosis in a later stage. These data provide a basis for the study of the response mechanism of fish to cold.
Asunto(s)
Sistema de Señalización de MAP Quinasas , Proteína Quinasa 8 Activada por Mitógenos , Animales , Proteína Quinasa 8 Activada por Mitógenos/genética , Proteína Quinasa 8 Activada por Mitógenos/metabolismo , Proteína Quinasa 9 Activada por Mitógenos/genética , Proteína Quinasa 9 Activada por Mitógenos/metabolismo , Proteína Quinasa 9 Activada por Mitógenos/farmacología , Fosforilación , Respuesta al Choque por Frío , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , ApoptosisRESUMEN
2-acetyl furanonaphthoquinone (FNQ) is a naturally occurring drug with enhanced toxicity versus glucose-starved tumor cells, which frequently show topoisomerase II drug resistance. Since loss of p53 tumor suppressor function or overexpression of the anti-apoptotic bcl-2 gene can decrease susceptibility to some cancer therapies, we now investigated the effect of FNQ against genetically matched C8161 melanoma cell lines transduced to express unequal levels of Bcl-2, or engineered to harbour a functional wt p53 for comparison with dominant-negative mutant p53 R175H. Cells with differing p53 genotype showed susceptibility to FNQ. However, this response was attenuated in those overexpressing mutant p53, although a brief p53 induction was early seen in FNQ-treated wt p53 cells. Cells susceptible to FNQ showed cleavage of anti-apoptotic Mcl-1, sustained activation of the c-Jun N-terminal Kinase (p-JNK), and apoptosis-associated PARP fragmentation, all of which were counteracted in bcl-2 overexpressing cells. Suppression of JNK activation with the specific inhibitor, SP600125 also prevented FNQ-mediated cell death. Our data suggests that Bcl-2, persistent JNK phosphorylation and cleavage of anti-apoptotic Mcl-1 are key events controlling susceptibility to FNQ.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Furanos/farmacología , Melanoma/metabolismo , Proteína Quinasa 9 Activada por Mitógenos/metabolismo , Naftoquinonas/farmacología , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Apoptosis/genética , Caspasas/genética , Caspasas/metabolismo , Caspasas/farmacología , Línea Celular Tumoral , Humanos , Melanoma/genética , Proteína Quinasa 9 Activada por Mitógenos/genética , Proteína Quinasa 9 Activada por Mitógenos/farmacología , Fosforilación/efectos de los fármacos , Proteínas Proto-Oncogénicas c-bcl-2/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/farmacología , Proteína X Asociada a bcl-2/genética , Proteína X Asociada a bcl-2/metabolismo , Proteína X Asociada a bcl-2/farmacologíaRESUMEN
Cells respond to a variety of extracellular and intracellular forms of stress by down-regulating rRNA synthesis. We have investigated the mechanism underlying stress-dependent inhibition of RNA polymerase I (Pol I) transcription and show that the Pol I-specific transcription factor TIF-IA is inactivated upon stress. Inactivation is due to phosphorylation of TIF-IA by c-Jun N-terminal kinase (JNK) at a single threonine residue (Thr 200). Phosphorylation at Thr 200 impairs the interaction of TIF-IA with Pol I and the TBP-containing factor TIF-IB/SL1, thereby abrogating initiation complex formation. Moreover, TIF-IA is translocated from the nucleolus into the nucleoplasm. Substitution of Thr 200 by valine as well as knock-out of Jnk2 prevent inactivation and translocation of TIF-IA, leading to stress-resistance of Pol I transcription. Our data identify TIF-IA as a downstream target of the JNK pathway and suggest a critical role of JNK2 to protect rRNA synthesis against the harmful consequences of cellular stress.
