BMP4 was required for the premature senescence induced by adriamycin in lung cancer cells. A, Western blotting confirmation of siRNA-mediated BMP4 knockdown. The negative control was an irrelevant siRNA. B, knockdown of BMP4 resumed the proliferation of NCI-H460 cells and A549 cells inhibited by adriamycin. Cells transfected with BMP4 siRNA vectors were treated with adriamycin for 3 days and cell proliferation was tested by MTT assay. ^*, p < 0.05; ^**, p < 0.01 versus untreated group. #, p < 0.05; ##, p < 0.01 versus adriamycin-treated group. Data are based on three independent experiments. C–E, BMP4 restrained cell senescence induced by adriamycin. NCI-H460 and A549 cells were transfected with BMP4 siRNA, and then treated with adriamycin at 500 nm and 2 μm, respectively. The senescence was examined by SA-β-gal activity assay, SA-β-gal staining, and immunofluorescence. ^*, p < 0.05; ^**, p < 0.01 versus untreated group. #, p < 0.05; ##, p < 0.01 versus adriamycin-treated group. Data are based on three independent experiments. F, Western blotting confirmation of BMP4 overexpression in the pSTAR-hBMP4 stably transfected cell line. pSTAR-hBMP4 cells were treated with DOX for 24 h, and Western blotting was carried out with antibody against BMP4. pSTAR cells treated with DOX was used as the control. G, Western blotting detection of BMP4 overexpression in pSTAR-hBMP4 cells treated with 10 μg/ml DOX for the indicated days. H, Western blotting analysis of the mount of BMP4 overexpression in pSTAR-hBMP4 cells treated with 10 μg/ml DOX, in comparison with that in NCI-H460 cells treated with adriamycin. I, BMP4 inhibited the proliferation of NCI-H460 cells as measured by MTT assay. pSTAR (left panel) and pSTAR-hBMP4 (right panel) cells were seeded on 96-well plates at a density of 2 × 10³ cells/well, respectively. After treatments with DOX for the times indicated, cell viability was assessed by measuring absorbance at 492 nm with a microplate reader. J and K, BMP4 induced premature senescence in NCI-H460 cells. SA-β-gal activity assay and SA-β-gal staining was performed on cells treated with DOX for 6 days. NCI-H460 cells treated with adriamycin were used as a positive control. ^*, p < 0.05; ^**, p < 0.01 (n = 3). L and M, BMP4 overexpression led to the appearance of heterochromatic DNA foci.

p16 and p21 were required for BMP4-induced premature senescence. A, BMP4 overexpression led to increases in p16 and p21 protein levels. Lysates were prepared 2 days following DOX treatment and probed with the indicated antibodies. β-Actin was used as an internal reference control. B, knockdown of BMP4 restrained the increased expression of p16 and p21 in response to adriamycin. NCI-H460 cells transfected with the BMP4 siRNA plasmid were treated with adriamycin, and then subjected to Western analysis with the p21 or p16 antibodies. C, Smad6 repressed BMP4-stimulated p16 and p21 expression. pSTAR-hBMP4 cells transfected with the Smad6-myc expression plasmid were treated with DOX, and then subjected to Western analysis with the p21 or p16 antibodies. D, Western blotting confirmation of p16 and p21 knockdown by p21 siRNA or p16 siRNA. The negative control was an irrelevant siRNA. E, knockdown of p16 and p21 resumed the proliferation of NCI-H460 cells inhibited by BMP4. pSTAR-hBMP4 cells transfected with p16 siRNA, p21 siRNA, or p16 siRNA plus p21 siRNA vectors were treated with DOX and cell proliferation was tested by MTT assay. ^*, p < 0.05; ^**, p < 0.01 versus untreated group. #, p < 0.05; ##, p < 0.01 versus DOX-treated group. Data are based on three independent experiments. F, knockdown of p16 and p21 inhibited premature senescence of NCI-H460 cells induced by BMP4. pSTAR-hBMP4 cells transfected with p16 siRNA, p21 siRNA, or p16 siRNA plus p21 siRNA vectors were treated with DOX and tested by SA-β-gal staining. G, BMP4 enhanced the endogenous SA-β-gal activity. An irrelevant control siRNA vector was used as the control. ^*, p < 0.05; ^**, p < 0.01 versus untreated group. #, p < 0.05; ##, p < 0.01 versus DOX. Data are based on three independent experiments. H, knockdown of p16 and p21 inhibited the formation of heterochromatin foci induced by BMP4. The average percentage was calculated based on three independent fields. ^*, p < 0.05; ^**, p < 0.01 versus untreated group. #, p < 0.05; ##, p < 0.01 versus DOX-treated group. Data are based on three independent experiments.

BMP4 overexpression increased the acetylation levels of histones H3 and H4 at p16 and p21 promoters. A and B, the acetylation levels of histones H3 and H4 were assessed by ChIP assays. pSTAR-hBMP4 cells were treated with DOX for 2 days and harvested for ChIP assay. Samples were immunoprecipitated (IP) with anti-Ac-H3 or Ac-H4 antibody, and the precipitated DNAs were amplified by using quantitative PCR. C, BMP4 overexpression enhanced the enrichment of p300 protein at the P16 and P21 promoters. pSTAR-hBMP4 cells were treated with DOX and ChIP assays were performed with anti-p300 antibody.

Adriamycin-induced premature senescence in company with the up-regulation of BMP4 expression in lung cancer cells. A, adriamycin (Adr) inhibited the proliferation of NCI-H460 and A549 cells as measured by MTT assays. NCI-H460 and A549 cells were treated with 500 nm and 2 μm adriamycin for 2 h, respectively, and cell viability was assessed by measuring absorbance at 492 nm with a microplate reader. B, exposure of NCI-H460 and A549 cells to adriamycin caused premature senescence. SA-β-gal staining was performed on cells cultured for 6 days after adriamycin treatment. C, adriamycin increased the endogenous SA-β-gal activity. The SA-β-gal activity assay was performed using o-nitrophenyl-d-galactopyranoside as substrate at pH 6.0 in NCI-H460 and A549 cells treated with adriamycin for 6 days. ^*, p < 0.05; ^**, p < 0.01 (n = 3). D, treatment of cells with adriamycin led to the appearance of heterochromatic DNA foci. NCI-H460 and A549 cells were treated with adriamycin at 500 nm and 2 μm for 2 h, respectively. Cells were stained with 4′,6-diamidino-2-phenylindole (DAPI), and heterochromatic foci were visualized by confocal microscopy. Immunostaining for tri-Me-K9-H3 showed its localization in discrete heterochromatic foci in adriamycin-treated cells. E, the average percentage of cells with heterochromatic foci (based on counting of 3 independent fields). ^*, p < 0.05; ^**, p < 0.01. F and G, changes of endogenous BMP4 and TGF-β1 mRNA levels in response to adriamycin stimulus were measured by RT-PCR and quantitative PCR. NCI-H460 and A549 cells were treated with adriamycin for 2 days, and BMP4 and TGF-β1 mRNA was measured by RT-PCR.β-actin was used as an internal control. H, Western blotting estimation of BMP4 protein levels in NCI-H460 and A549 cells treated with adriamycin.

Senescence triggered by BMP4 was dependent upon the Smad signaling pathway. A, Western blotting confirmation of the ectopic expression of Smad6 in NCI-H460 cells. B, Smad6 restrained cellular senescence induced by BMP4. pSTAR-hBMP4 cells transfected with Smad6-myc expression plasmid were exposed to DOX for 6 days and tested for the SA-β-gal activity. The pcDNA3.1 empty vector was used as a negative control. ^*, p < 0.05 versus untreated group. #, p < 0.05, versus the DOX-treated group. Data are based on three independent experiments. C, representative photomicrographs of the SA-β-gal staining. D, the average percentages of cells containing heterochromatin foci were greatly decreased upon Smad6 overexpression. E–G, Smad6 repressed cellular senescence induced by adriamycin as judged by SA-β-gal activity assays, SA-β-gal staining, and immunofluorescence. ^*, p < 0.05; ^**, p < 0.01 versus untreated group. #, p < 0.05; ##, p < 0.01 versus adriamycin-treated group. Data are based on three independent experiments. H, BMP4 elevated the phosphorylation levels of the downstream proteins Smad1/5/8. Western blotting estimation of the phosphorylation levels of Smad1/5/8 in pSTAR-hBMP4 cells treated with DOX.

BMP4 induced transcription of p16 and p21 by increasing Smad1/5/8 transcriptional activity. A, BMP4 up-regulated the endogenous P16 and P21 mRNA levels in NCI-H460 cells. pSTAR-hBMP4 cells were treated with DOX for 2 days, and P16 or P21 mRNA was measured by RT-PCR. B, quantitative estimation of P16 and P21 mRNA using real-time PCR. C, BMP4 up-regulated P16 and P21 promoter activities. 1 μg of the pEGFP-hBMP4 expression plasmid, together with 1 μg of P16 or P21 reporter plasmid were cotransfected into NCI-H460 cells. Luciferase activity was determined 30 h after transfection and normalized to the Renilla activity. The pcDNA3.1 plasmid was used as a negative control. ^*, p < 0.05; ^**, p < 0.01 (n = 3). D, diagrams of the 5′-flanking regions of P16 and P21 genes. Lines below (P1–P3) and (Q1–Q3) indicate the three regions of the P16 and P21 promoter amplified in ChIP assays, respectively. E, BMP4 overexpression potentiated the binding of phospho-Smad1/5/8 at different regions of P16 and P21 promoters, as revealed by ChIP assays. pSTAR-hBMP4 cells were treated with DOX for 2 days and harvested for ChIP assay. Samples were immunoprecipitated with anti-phospho-Smad1/5/8 antibodies. Precipitated DNAs were amplified by using quantitative PCR.

p300 was recruited to P16 and P21 promoters by transcription factors Smad1/5/8. A, the binding abilities of Smad1/5/8 were declined upon Smad6 overexpression. pSTAR-hBMP4 cells were transfected with Smad6 expression plasmid and treated with DOX before being subjected to ChIP assays. Samples were immunoprecipitated (IP) with anti-phospho-Smad1/5/8 antibodies and amplified by quantitative PCR. B, the enrichments of p300 at different regions of P16 and P21 promoters were significantly decreased when the binding abilities of Smad1/5/8 were restrained by Smad6 overexpression. ChIP assays were performed with antibody against p300. ^*, p < 0.05 versus untreated group. #, p < 0.05 versus DOX-treated group. Data are from three independent experiments. C and D, ChIP assays for detection of the presence of acetylated histones H3 and H4 at p16 and p21 promoters in pSTAR-hBMP4 cells transfected with Smad6 expression plasmid. ^*, p < 0.05 versus untreated group. #, p < 0.05 versus DOX-treated group. Data are from three independent experiments.