Manipulation of redox signaling in mammalian cells enabled by controlled photogeneration of reactive oxygen species.
Reactive oxygen species (ROS) comprise a group of noxious byproducts of oxidative processes which participate in the induction of many common diseases. However, understanding their role in the regulation of normal physiological redox signaling is currently evolving. Detailed study of the dynamic functions of ROS within the biological milieu is difficult because of their high chemical reactivity, short lifetime, minute concentrations and cytotoxicity at high concentrations. In this study, we show that increasing intracellular ROS levels, set off by controlled in situ photogeneration of a nontoxic bacteriochlorophyll-based sensitizer initiate responses in cultured melanoma cells. Using hydroethidine as detector, we determined light-dependent generation of superoxide and hydroxyl radicals in cell-free and cell culture models. Monitoring the ROS-induced responses revealed individual and differential behavior of protein kinases [p38, mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and Akt] as well as effects on the subcellular distribution of phosphorylated p38. Furthermore, alterations in morphology and motility and effects on cell viability as a function of time and photosensitizer doses were observed. Following mild ROS challenge, enzymatic and cellular changes were observed in the majority of the cells, without inducing extensive cell death. However, upon vigorous ROS challenge, a similar profile of the overall responses was observed, terminating in cell death. This study shows that precisely controlled photogeneration of ROS can provide simple, fine-tuned, noninvasive manipulation of ROS-sensitive cellular responses ranging from individual enzymes to gross behavior of target cells. The observations made with this tool enable a dynamic and causal correlation, presenting a new alternative for studying the role of ROS in cellular redox signaling.
Posen
Yehudit
Y
Department of Biological Regulation, Weizmann Institute of Science, PO Box 26, Rehovot 76100, Israel.
Kalchenko
Vyacheslav
V
Seger
Rony
R
Brandis
Alexander
A
Scherz
Avigdor
A
Salomon
Yoram
Y
D000818:Animals; D001429:Bacteriochlorophylls / Q000737:chemistry; D045744:Cell Line, Tumor; D002470:Cell Survival; D002474:Cell-Free System; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D005434:Flow Cytometry; D006861:Hydrogen Peroxide / Q000494:pharmacology; D007091:Image Processing, Computer-Assisted; D047468:Immunoprecipitation; D007501:Iron / Q000494:pharmacology; D048031:JNK Mitogen-Activated Protein Kinases / Q000378:metabolism; D008027:Light; D020935:MAP Kinase Signaling System; D008545:Melanoma / Q000378:metabolism; D051379:Mice; D008856:Microscopy, Fluorescence; D018715:Microscopy, Video; D009499:Neutral Red / Q000494:pharmacology; D010084:Oxidation-Reduction; D010100:Oxygen / Q000737:chemistry / Q000378:metabolism; D010617:Phenanthridines / Q000737:chemistry / Q000494:pharmacology; D010766:Phosphorylation; D010778:Photochemotherapy; D011419:Propidium / Q000494:pharmacology; D011485:Protein Binding; D017382:Reactive Oxygen Species; D012694:Serine / Q000737:chemistry; D015398:Signal Transduction; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D013997:Time Factors; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
D001429:Bacteriochlorophylls; C045076:Fenton's reagent; D010617:Phenanthridines; D017382:Reactive Oxygen Species; D009499:Neutral Red; D011419:Propidium; C058428:hydroethidine; D012694:Serine; D006861:Hydrogen Peroxide; D007501:Iron; D048049:Extracellular Signal-Regulated MAP Kinases; D048031:JNK Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D010100:Oxygen