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First published online 19 April 2005
doi: 10.1242/jcs.02323

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Manipulation of redox signaling in mammalian cells enabled by controlled photogeneration of reactive oxygen species

¹ Department of Biological Regulation, Weizmann Institute of Science, PO Box 26, Rehovot 76100, Israel
² Department of Plant Sciences, Weizmann Institute of Science, POB 26, Rehovot 76100, Israel

View larger version (22K): [in a new window]   Fig. 1. Photosensitization of Pd-Bchl-Ser: effect on cell survival. M2R cells (4x104 cell/well) grown in 96-well plates were preincubated with increasing concentrations of Pd-Bchl-Ser, and either illuminated (light) or not illuminated (dark). Cells were then reincubated for 24 hours in the culture incubator and cell survival was determined using the neutral red assay. Points represent means of triplicate determinations ± s.d.

View larger version (37K): [in a new window]   Fig. 2. Induction of ROS generation within cells upon photosensitization with Pd-Bchl-Ser and light. Cell-free spectroscopic analysis (A) 2.5 µM Pd-Bchl-Ser were incubated with 50 µM HE, 220 µg DNA and 100 µM DTPA in 1 ml PBS (open triangles) and then illuminated for 5 minutes at 20 mW/cm2. Controls: dark control (closed circles) was not illuminated; illuminated samples with SOD (6x103 U; closed diamonds). Basic fluorescence in the illuminated sample was determined in the absence of DNA (open squares), or ethidium standard containing DNA and 60 µM ethidium bromide (solid line). Fluorescence intensity was then determined upon excitation at 480 nm with a slit width of 4 nm and an emission slit width of 8 nm. (B) As in A, but samples were prepared in 1.5% Triton X-100 and an additional control containing 8 mM melatonin was included (open circles). When added, SOD concentration was 100 U/ml (closed diamonds). (C) Fenton reaction: 1 mM H2O2 was incubated with FeSO4 at a 1:1 molar ratio in the presence of 50 µM HE and 220 µg DNA in a final volume of 1 ml PBS (closed diamonds) and fluorescence was determined as in A. Controls: reaction mixture with 1 mM H2O2 and 1 mM FeSO4 in the presence of 24 mM melatonin (closed circles), with 3 mM H2O2 without FeSO4 (closed triangles), with 3 mM FeSO4 without H2O2 (crosses) or with HE alone (open squares), ethidium bromide and DNA as in A. (D) Oxidation of HE in cultured cells. M2R cells were preincubated with 100 nM Pd-Bchl-Ser, rinsed and medium replaced with 10 µM HE in PBS. Cells were illuminated immediately thereafter for 0.5 (open diamonds), 1 (open triangles), 2 (closed diamonds), 5 (open circles) or 10 (open squares) minutes and collected for determination of fluorescence emission. Dark control was treated with HE for 10 minutes in the dark (closed circles). Graph insert illustrates fluorescence maxima at 580 nm as a function of time. (E) M2R cell monolayers were preincubated with 0, 15, 30, 60 or 100 nM Pd-Bchl-Ser for 4 hours and then treated with 2 µM HE for 10 minutes and illuminated prior to FACS analysis. Bars represent the percentage of cells emitting fluorescence following treatment and the graph represents the fluorescence intensity. (F) M2R cells were preincubated with 0, 15, 30, 60 or 100 nM Pd-Bchl-Ser for 4 hours and then treated with 10 µM H2DCFDA for 10 minutes and illuminated prior to FACS analysis.

View larger version (96K): [in a new window]   Fig. 3. Commitment to death after treatment with Pd-Bchl-Ser and light. M2R cells were preincubated with Pd-Bchl-Ser at LD90 (DC) and then illuminated, reincubated (0 minutes, 45 minutes and 24 hours) and then stained with PI. Time points indicate the incubation time following illumination excluding the 15 minutes staining process. Phase-contrast microscopy is presented in the left column and fluorescence microscopy with PI staining is presented in the right column.

View larger version (65K): [in a new window]   Fig. 4. Morphological changes in M2R cells following treatment with Pd-Bchl-Ser and light. (A) M2R cells were treated at LD90 with no illumination (dark controls), LD90 and illumination (LD90) or LD20 and illumination (LD20) and selected groups of cells were continuously monitored over a 2.5-hour period using video microscopy. Videos were processed and the still photographs of indicated times are presented. By following individual cells throughout the frames, one can observe cell motility by following the morphological changes of particular cells over time. (B) For each plot, the paths of ten cells were measured, normalized and converted to wind rose display where all cells start from (0,0) in order to illustrate the average motility. UN, untreated; DC, dark control; LC, light control; LD20, LD20 treatment. Total cell displacement was calculated by measuring the change in position of the cell centroid every 30 minutes over a 2.5-hour period. An average of total cell displacement (D) ± s.e.m. for 10 randomly chosen cells in each group was calculated and is presented in the respective rose wind plot. Total displacement of LD20-treated cells in comparison to other experimental groups were significantly different (P values <0.0005). (C) Cell circularity was computed as circularity=4 x (area)/perimeter2. Normalized average ± s.e.m. of circularity of 10 randomly chosen individual cells in each group were plotted. Untreated (closed squares), dark control (open triangles), light control (closed circles), LD20 treatment (closed diamonds).

View larger version (20K): [in a new window]   Fig. 5. Phosphorylation and stimulation of catalytic activity of cellular p38 following photosensitization with Pd-Bchl-Ser. M2R cells were preincubated with Pd-Bchl-Ser at LD90 and illuminated, and cell lysates were prepared immediately thereafter. (A) Proteins (30 µg protein/lane) were separated by 10% SDS-PAGE and detected with anti-phosphorylated-p38 antibody by immunoblotting. (B) Immunoprecipitation of p38 with anti-phosphorylated p38 was performed using the respective cell lysates. Catalytic activity of p38 was assayed using myelin basic protein (MBP) and [-32P]ATP as substrates. Lysates were analyzed by autoradiography following SDS-PAGE. Detected bands show 32P coinciding with position of MBP.

View larger version (28K): [in a new window]   Fig. 6. Phosphorylation of protein kinases following treatment with Pd-Bchl-Ser at LD90 and light. M2R cell lysates were prepared at the indicated times following treatment. Proteins (75 µg protein/lane) were separated on 10% SDS-PAGE, blotted and immunodetected with anti-phosphorylated p38, ERK, JNK or Akt. Resting levels of phosphorylated protein kinases were determined on untreated (0 minutes) cells. Dark controls (DC) were treated with sensitizer but not illuminated. Blots reacted with anti-p38 and anti-ERK measured total amounts of protein blotted. Graphs represent relative phosphorylation of proteins as a percentage of the band density displaying the highest value on the given blot. The gray area denotes the duration of the illumination.

View larger version (33K): [in a new window]   Fig. 7. Phosphorylation of protein kinases following treatment with light and increasing concentrations of Pd-Bchl-Ser. Cells were preincubated with increasing concentrations of Pd-Bchl-Ser, illuminated for 10 minutes and reincubated for 45 minutes. Cell lysates were prepared and proteins were separated on 10% SDS-PAGE, blotted and immunodetected with anti-phosphorylated p38, ERK, JNK or Akt. Phosphorylation is presented as a percentage of the band density displaying the highest value on the respective blot.

View larger version (34K): [in a new window]   Fig. 8. Phosphorylation of protein kinases following treatment with Pd-Bchl-Ser at LD20 and light. M2R cell lysates were prepared at the indicated times following treatment. Proteins (75 µg protein/lane) were separated on 10% SDS-PAGE, blotted and immunodetected with anti-phosphorylated p38 or ERK. Resting levels of phosphorylated protein kinases were determined on untreated (0 minutes) cells. Blots reacted with anti-p38 and anti-ERK measured total amounts of protein blotted. The graph represents relative phosphorylation of proteins as percent of the band density displaying the highest value on the given blot. The gray area denotes the illumination time interval.

View larger version (121K): [in a new window]   Fig. 9. Cellular location of p38 following treatment of M2R cells with Pd-Bchl-Ser and light. (A) Cells were treated with Pd-Bchl-Ser at LD90 and light. Samples were fixed and permeabilized at varying time intervals after illumination (0-45 minutes) and stained with anti-phosphorylated p38. UN, untreated cells; UN-p38, untreated cells stained with anti-p38; anti-p-p38+dP peptide, immunostaining of cells treated with anti-phosphorylated p38 pre-reacted with its immunogen, the doubly phosphorylated (dP) p38 peptide, was completely blocked demonstrating specificity of the staining. (B) As in A but cells were treated with Pd-Bchl-Ser at LD20 and light.

View larger version (29K): [in a new window]   Fig. 10. Correlation between photosensitizer concentrations, ROS levels and respective cellular responses: The data presented in Fig. 2E,F (ROS production DCF, HE), Fig. 7 (phosphorylation levels of Akt, p38, ERK, JNK) and Fig. 1 (cell death) were converted to percentages of maximal effect, and expressed as a function of Pd-Bchl-Ser concentration.