Article Figures & Data
Figures
- Figure 1.
Kir2.2 as a senescence-associated molecular marker. A, RT-PCR analysis of Kir2.2 in doxorubicin (10 nmol/L)–treated PC-3 cells. B, induction of senescence by low-dose (10 nmol/L) doxorubicin treatment. Left, expression of Kir2.2 in PC-3 cells after transfection of Kir2.2-GFP, as determined by Western blot analysis. Right, after culturing with doxorubicin for 5 d, the cells were stained for SA-β-Gal. The number on the right indicates the percentage of SA-β-Gal–positive cells. C, effects of siKir2.2 on the growth of PC-3 cells. a, expression of Kir2.2 in PC-3 cells after transfection of siRNAs examined by RT-PCR and Western blot analysis. b, siKir2.2 concentration-dependent inhibition of PC-3 cell growth. Cell percentage: viable cell number in test sample/viable cell number in control sample (PBS), 100%. Effects of siKir2.2 (30 nmol/L) versus siC (30 nmol/L) on cell growth kinetics (c) and long-term colony formation (d). The values represent the means ± SDs (columns) of three independent experiments (P, siKir2.2 versus siC). D, exogenous expression of Kir2.2 (pKir2.2) rescued cells from siKir2.2-induced senescence. The cells were stained for SA-β-Gal activity 5 d after transfection.
- Figure 2.
Kir2.2 knockdown induces cell cycle arrest but not cell death. PC-3 cells transfected with siC or siKir2.2 (30 nmol/L) were collected 5 d after transfection and subjected to a flow cytometric analysis. A, effect of Kir2.2 knockdown on the cell cycle distribution of PC-3 cells. The results of one representative experiment are presented. The values shown represent the mean ± SD (columns) of three independent experiments. B, changes in the expression of cell cycle–related proteins following siRNA transfection. PC-3 cells were seeded at a concentration of 2 × 105/mL before siRNA transfection and then harvested at the indicated times. Cell lysates containing 20 μg of protein were analyzed by SDS-PAGE/Western blotting using the antibodies shown on the right. Bottom, bands on Western blots were quantified by densitometry, and the information is presented in histogram format. Data represent means ± SDs from three independent experiments. C, flow cytometry following Annexin V and propidium iodide staining. D, the percentage of terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling–positive cells was determined using flow cytometry. PBS and etoposide (1 μg/mL for 48 h) were used as negative and positive controls, respectively.
- Figure 3.
p27 is required for Kir2.2 knockdown-induced senescence. A, Western blot analysis of lysates from PC-3 cells transfected with the indicated siRNAs. The day after transfection, cells were analyzed for p27 expression by Western blotting. B, knockdown of p27 dramatically decreased the percentage of senescent cells induced by Kir2.2 knockdown from 83 ± 3% to 12 ± 4% (P < 0.0001). Cells were scored for SA-β-Gal-positivity (senescence) 5 d after transfection with siRNA. C, effect of Kir2.2 expression on the cell cycle of PC-3 cells. Cells were treated with doxorubicin (10 nmol/L) for 5 d and then fixed with 70% ethanol and incubated with RNase A and the DNA-intercalating dye propidium iodide. Results of a representative experiment of three independent experiments are presented.
- Figure 4.
siKir2.2 induces senescence in multiple cancer cell lines. A, PC-3, LNCaP, SNU638, and MCF7 cells were stained for SA-β-Gal 5 d after transfection with siRNAs (50 nmol/L). The number indicates the percentage of SA-β-Gal–positive cells. B, RT-PCR analysis of senescence-associated genes in multiple cancer cells using primers described previously (20). C, induction of senescence-associated markers after transfection of PC-3 cells with the indicated siRNAs (20 nmol/L). siC, siControl; siK, siKir2.2. a, mitochondrial dysfunction. Top left, fluorescence of cells on flow cytometry after staining with Mitotracker Red (Invitrogen, M7512), which permits estimation of mitochondrial mass within cells. Top right, MitoSox fluorescence. MitoSox is an indicator of mitochondrial superoxide level and therefore a measure of mitochondrial ROS. Bottom left, JC-1 fluorescence. An increase in green fluorescence indicates mitochondrial membrane depolarization. Bottom right, ATP content in whole cells. b, immunofluorescence of γH2AX, SAHF, or PML bodies. c, estimation of IL-6 and IL-8 levels in the medium. An ELISA kit (R&D Systems) was used; data were normalized to cell number (pg secreted protein per cell per day). d, Significant inactivation of phosphatase. Top, PP1/2A activity. Bottom, alkaline phosphatase activity. The data were normalized to protein amount (pmol phosphate/μg protein/min). *, P < 0.05; comparison between siC and siKir2.2 cells.
- Figure 5.
Kir2.2 knockdown induces senescence by increasing ROS accumulation in PC-3 cells. A, Kir2.2 knockdown induced an increase in ROS accumulation. DCF fluorescence (fold) indicates ROS generation. B, the antioxidant NAC (5 mmol/L) blocked siKir2.2-induced senescence. PC-3 cells were stained for SA-β-Gal 5 d after the transfection of siRNAs (50 nmol/L). The number indicates the percentage of SA-β-Gal–positive cells. C, p27 is required for Kir2.2 knockdown-induced ROS generation, and p27 is not synthesized in the absence of ROS, under the same conditions. Western blot analysis of lysates from PC-3 cells transfected with the indicated siRNAs with or without NAC (5 mmol/L). The day after transfection, cells were analyzed for p27 and Kir2.2 expression by Western blotting. D, knockdown of p27 dramatically decreased ROS generation induced by Kir2.2 knockdown (P = 0.0001). ROS levels were analyzed using the fluorescent dye DCF-DA 5 d after transfection with siRNA.
- Figure 6.
Kir2.2 knockdown decreases in vivo tumorigenesis. A, siRNA (50 nmol/L) against Kir2.2 suppresses tumor growth in nude mice. For each injection, 1.5 × 106 PC-3 cells were transfected with siC or siKir2.2 and implanted s.c. into the flanks of 5-wk-old female athymic nu/nu mice. Top, Western blot shows Kir2.2 expression in representative samples. Bottom, gross tumors representative of groups immediately after resection. Day 41 siKir2.2 xenografts were strikingly smaller than siC xenografts (P < 0.0001, n = 10 mice per group; t test). Points, SEM. B, effect of intratumoral siKir2.2 injection on the growth of established tumors. When tumors reached an average size of 40 to 50 mm3 (∼4 wk), the mice received two times (days 1 and 7) of intratumoral injections of siRNA as a mixture of siRNA (50 nmol/L) in 100 μL of Effectene per injection (n = 10). Points, SEM. The top Western blot shows Kir2.2 expression levels in representative samples 2 d after siRNA injection. C, immunohistochemical staining of sections from formalin-fixed, paraffin-embedded tumor samples (resected on postimplantation day 41) showed intense p27 and PAI-1 immunoreactivity, but reduced Ki67 staining, in cells from siKir2.2-transfected tumors. SA-β-Gal staining of fresh tumor tissue 5 d after siRNA injection revealed Kir2.2 knockdown-induced senescence.
Additional Files
Supplementary Data, Lee, et al.
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Volume 9, Issue 11
