RESUMO
BACKGROUND: Genetically encoded photosensitizers are a promising optogenetic instrument for light-induced production of reactive oxygen species in desired locations within cells in vitro or whole body in vivo. Only two such photosensitizers are currently known, GFP-like protein KillerRed and FMN-binding protein miniSOG. In this work we studied phototoxic effects of miniSOG in cancer cells. METHODS: HeLa Kyoto cell lines stably expressing miniSOG in different localizations, namely, plasma membrane, mitochondria or chromatin (fused with histone H2B) were created. Phototoxicity of miniSOG was tested on the cells in vitro and tumor xenografts in vivo. RESULTS: Blue light induced pronounced cell death in all three cell lines in a dose-dependent manner. Caspase 3 activation was characteristic of illuminated cells with mitochondria- and chromatin-localized miniSOG, but not with miniSOG in the plasma membrane. In addition, H2B-miniSOG-expressing cells demonstrated light-induced activation of DNA repair machinery, which indicates massive damage of genomic DNA. In contrast to these in vitro data, no detectable phototoxicity was observed on tumor xenografts with HeLa Kyoto cell lines expressing mitochondria- or chromatin-localized miniSOG. CONCLUSIONS: miniSOG is an excellent genetically encoded photosensitizer for mammalian cells in vitro, but it is inferior to KillerRed in the HeLa tumor. GENERAL SIGNIFICANCE: This is the first study to assess phototoxicity of miniSOG in cancer cells. The results suggest an effective ontogenetic tool and may be of interest for molecular and cell biology and biomedical applications.
Assuntos
Flavoproteínas/genética , Terapia Genética/métodos , Oxigênio/metabolismo , Fármacos Fotossensibilizantes/metabolismo , Animais , Caspase 3/genética , Caspase 3/metabolismo , Morte Celular/genética , Linhagem Celular , Linhagem Celular Tumoral , Membrana Celular/genética , Membrana Celular/metabolismo , Cromatina/genética , Cromatina/metabolismo , Dano ao DNA , Reparo do DNA , Dermatite Fototóxica/etiologia , Dermatite Fototóxica/genética , Dermatite Fototóxica/metabolismo , Feminino , Flavoproteínas/metabolismo , Células HEK293 , Células HeLa , Humanos , Luz/efeitos adversos , Camundongos , Camundongos Nus , Mitocôndrias/genética , Mitocôndrias/metabolismo , Riboflavina/genética , Riboflavina/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Alternative splicing plays a major role in increasing proteome complexity and regulating gene expression. Here, we developed a new fluorescent protein-based approach to quantitatively analyze the alternative splicing of a target cassette exon (skipping or inclusion), which results in an open-reading frame shift. A fragment of a gene of interest is cloned between red and green fluorescent protein (RFP and GFP)-encoding sequences in such a way that translation of the normally spliced full-length transcript results in expression of both RFP and GFP. In contrast, alternative exon skipping results in the synthesis of RFP only. Green and red fluorescence intensities can be used to estimate the proportions of normal and alternative transcripts in each cell. The new method was successfully tested for human PIG3 (p53-inducible gene 3) cassette exon 4. Expected pattern of alternative splicing of PIG3 minigene was observed, including previously characterized effects of UV light irradiation and specific mutations. Interestingly, we observed a broad distribution of normal to alternative transcript ratio in individual cells with at least two distinct populations with â¼45% and >95% alternative transcript. We believe that this method is useful for fluorescence-based quantitative analysis of alternative splicing of target genes in a variety of biological models.
Assuntos
Processamento Alternativo , Éxons , Corantes Fluorescentes , Proteínas de Fluorescência Verde , Proteínas Luminescentes , Citometria de Fluxo , Genes Reporter , Proteínas de Fluorescência Verde/genética , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas Luminescentes/genética , Microscopia de Fluorescência , Proteínas Proto-Oncogênicas/genética , Análise de Célula Única , Proteína Vermelha FluorescenteRESUMO
We developed a genetically encoded, highly specific fluorescent probe for detecting hydrogen peroxide (H(2)O(2)) inside living cells. This probe, named HyPer, consists of circularly permuted yellow fluorescent protein (cpYFP) inserted into the regulatory domain of the prokaryotic H(2)O(2)-sensing protein, OxyR. Using HyPer we monitored H(2)O(2) production at the single-cell level in the cytoplasm and mitochondria of HeLa cells treated with Apo2L/TRAIL. We found that an increase in H(2)O(2) occurs in the cytoplasm in parallel with a drop in the mitochondrial transmembrane potential (DeltaPsi) and a change in cell shape. We also observed local bursts in mitochondrial H(2)O(2) production during DeltaPsi oscillations in apoptotic HeLa cells. Moreover, sensitivity of the probe was sufficient to observe H(2)O(2) increase upon physiological stimulation. Using HyPer we detected temporal increase in H(2)O(2) in the cytoplasm of PC-12 cells stimulated with nerve growth factor.