Spatial Distribution of Protein Damage by Singlet Oxygen in Keratinocytes

Abstract

Singlet oxygen may be generated in cells by either endogenous or exogenous photosensitizers as a result of exposure to UV or visible irradiation. We have used immuno-spin trapping (Free Rad. Biol. Med. 36: 1214 to identify the subcellular targets of singlet oxygen generated by Rose Bengal (RB). Confocal fluorescence microscopy of HaCaT keratinocytes incubated with RB clearly showed that the dye entered the cells and was located mainly in the perinuclear region, probably associated with the Golgi apparatus and endoplasmic reticulum. Previous studies by Wright and coworkers (Free Rad. Biol. Med. 34, 637, 2003) have shown that long lived protein hydroperoxides (POOH) are present in cells exposed to singlet oxygen generating dyes. The addition of reducing metal ions such as Cu + to POOH results in the generation of protein derived radicals, POO · and PO · , which react with the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) to give relatively stable spin adducts. In order to determine the subcellular localization of the protein-DMPO adducts, we exposed keratinocytes to RB/light exposure and then incubated the cells with Cu + and DMPO. After staining with antibody against DMPO followed by a secondary Alexa Fluor 488 goat anti-rabbit IgG, the intracellular distribution of protein-DMPO adducts was determined by confocal microscopy. The subcellular localization of the protein DMPO adducts was coincident with that of Rose Bengal. This approach may provide information on the spatial distribution of singlet oxygen generated in cells.

Immunosuppressive Cyclosporin A Activates AKT in Keratinocytes through PTEN Suppression

“…siRNA and shRNA Transfection-Transfection of cells with siRNA or shRNA was performed as described previously (16,19). An ON-TARGETplus SMARTpool siRNA targeting PTEN (Dharmacon, L-003023) was used, which included four duplexes.…”

Anthracene-based fluorescent nanoprobes for singlet oxygen detection in biological media

“…The green volume is the cytoplasm and the red dots represent the nanoADPA aggregates.In a second series of experiments, HeLa cells were co-incubated with nanoADPA and Rose Bengal (RB), an excellent 1 O 2 photosensitizer. RB is cell-permeable, can be selectively excited where ADPA does not absorb (561 nm) and shows very low absorption where ADPA emits [49,50]. Moreover, we could find no evidence for interaction between nanoADPA and RB in PBS or in DMEM (Figure S12).Figure 6shows that the intracellular fluorescence of nanoADPA decreased to 1/3rd of its original value when the cells were irradiated at 561 nm for 10 minutes, while no-fluorescence decrease could be observed in the absence of RB.…”

Effects of UV-A LED light irradiation on growth of cultured RAW 264.7 cells

“…Singlet oxygen also oxidized cholesterol of the mammalian plasma membrane (Korytowski, Schmitt, and Girotti 2010). The addition of reducing metal ions, including Fe 2C and Cu C to POO and P resulted in generation of proteinderived radicals (He et al 2008). In the present study, because of the presence of serum protein, lipids, and small amounts of metal ions, these molecules, as suggested by He et al (2008), may have been peroxidized and radicals were formed in cells or plasma membrane components (proteins and lipids) due to singlet oxygen induced by UV-A irradiation.…”

“…The addition of reducing metal ions, including Fe 2C and Cu C to POO and P resulted in generation of proteinderived radicals (He et al 2008). In the present study, because of the presence of serum protein, lipids, and small amounts of metal ions, these molecules, as suggested by He et al (2008), may have been peroxidized and radicals were formed in cells or plasma membrane components (proteins and lipids) due to singlet oxygen induced by UV-A irradiation. However, in the UV-A irradiated culture medium, other signals, besides DMPO-OH, were not detected (Figure 7).…”