Abstract
Cell senescence, an irreversible cell cycle arrest, reflects a safeguard program that limits the capacity of uncontrolled cell proliferation. Treatment of tumor cells with certain chemotherapeutic agents activates premature senescence to decrease the tumorigenecity. Here we show that sublethal concentrations of adriamycin could induce premature senescence in lung cancer cells. Adriamycin treatment resulted in the up-regulation of BMP4, which is underexpressed in NSCLC (non-small cell lung cancers). Moreover, the BMP4-Smad pathway played a key role in mediating adriamycin-induced senescence. Overexpression of BMP4 was able to induce premature senescence in lung cancer cells and this process required the participation of cyclin/cyclin-dependent kinase (cdk) inhibitors p16(INK4a) and p21(WAF1/cip1). We also show that increases of p16(INK4a) and p21(WAF1/cip1) expression in response to BMP4 were mediated by the Smad signaling pathway. Furthermore, our data revealed that p300 was recruited to P16(INK4a) and P21(WAF1/cip1) promoters by Smad1/5/8 to induce the hyperacetylation of histones H3 and H4 at the promoters. The present study provides useful clues to the evaluation of the potentiality of BMP4 as a responsive molecular target for cancer chemotherapy.
Highlights
High acidic -galactosidase (SA--gal)3 activity, and it usually occurs within a week of exposure to sublethal stresses
Adriamycin-induced Premature Senescence in Company with the Up-regulation of BMP4 in Lung Cancer Cells—Because growth arrest is a necessary step leading to cell senescence, we first determined whether adriamycin treatment affected cell proliferation
These results indicated that adriamycin could induce tumor cell senescence to impair the tumorigenecity in non-small cell lung cancer cells
Summary
Plasmid Constructs—The P16INK4a (hereafter P16) promoter reporter (Ϫ869 to ϩ1 bp from the cap site) ligated to the luciferase reporter gene (pGL2 basic, Promega) was provided by Dr E. The pSTAR was constructed with neomycin resistance for selection of stably transfected cells, and a cloning cassette for placing a gene of interest under the control of the tetO DNA motif. This plasmid expresses rtTAnls, which upon association with tetracycline, binds to and drives gene expression from the tetO DNA motif. The cell line that induceably expresses BMP4 protein was designated the pSTAR-hBMP4. Real-time PCR was performed on an ABI Prism 7000 Sequence Detection System (Applied Biosystems), following the manufacturer’s protocol, with SYBR Green (TaKaRa, Osaka, Japan) used as a double-stranded DNA-specific fluorescent dye. The primer pairs for BMP4 gene were: sense, 5Ј-agcatgtcaggattagccga-3Ј and antisense, 5Ј-tggagatggcactcagttca-3Ј. The P53-specific siRNA sequence was 5Ј-gactccagtggtaatctac-3Ј [32]
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