Comparative analysis of p14ARF transcriptional and post-transcriptional regulation of cyclin D1, p53 and p21 by p14ARF.

<p>MCF-7p14ARF 4a cells were treated with IPTG (lanes, 2, 4, 5, 8) or control (lanes 1, 3, 5, 7). Cells were subsequently treated with CHX, Act D, MG132 or vehicle (control). <b>A.</b> Cyclin D1, p21 and p53 protein expression was analysed by Western blot, post-treatments as indicated. <i>Column graph:</i> Protein expression was analysed by densitometry and expressed as arbitrary units (AU). <b>B.</b> Time course of p53, cyclin D1 expression in IPTG induced MCF-7p14ARF cells with MG132, CHX and Act D. Representative Western blot from three experiments show relative protein expression of p14ARF, p53 and cyclin D1. C. Line graph of % change in cyclin D1 and p53 protein expression when compared to control post MG132 and CHX treatments.</p

P14ARF reactivates the p53 pathway and increases cyclin D1 protein in MCF-7 cells.

<p><b>A.</b> MCF-7p14ARF clonal cell lines (12, 4a, 8a and 9a) were induced with IPTG and levels of p14ARF were analysed by Western blot. β-actin was used as a loading control. The subcellular distribution of p14ARF (red staining) was analysed by immunofluorescence staining. Nuclei were stained with Hoechst 33342 (blue). <b>B.</b> Cells were grown on cover slips in 6-well plates and EDU was added directly to the cell culture medium 6h and 15h post-IPTG (5 mM) treatment and cells were incubated for a further 20h. Images of individual cells undergoing DNA synthesis within this 20h period were captured. Proliferating cells were visualised using Alexa 488 (green) and nuclei using Hoechst (blue). Column graph: % EDU stained cells (+/− SD). <b>C.</b> MCF-7p14ARF, MCF-7 parent +/− IPTG, were harvested over a time period of 24h and cell cycle phases analysed by flow cytometry. <b>D.</b> 24h +/− IPTG, sample proteins were analysed by Western blot. Relative expression of p53, p21, cyclin D1, total Rb and pRb phosphorylated at Ser⁷⁸⁰ [pRb (Ser⁷⁸⁰)], and p14ARF are shown. <b>E.</b> MCF-7p14ARF, clone 4a cells were treated +/− IPTG for 5 days and stained with SA-β-gal to detect senescence.</p

Regulation of <i>cyclin D1, p53, and p21</i> mRNA and protein expression of in MCF-7p14ARF.

<p>Cells were treated with IPTG, ICI 182780 (ICI), or E2 for 12 h. Top panel: relative gene expression of <b>A... </b><i>CCDN1</i>, <b>B... </b><i>TP53</i> and <b>C... </b><i>CDKN1A</i> mRNA quantified by qRT-PCR and relative expression normalised to control <i>ACTB. Columns,</i> Data represents the mean ± SEM of three separate experiments (** P<0.001. number = 9). Bottom panel: representative Western blots of <b>A.</b> cyclin D1, <b>B.</b> p53, and <b>C.</b> p21. Relative levels of protein are shown above each lane. β-actin was used as a control. The representative images are all from the same sample set, run on the same gel and Western blot.</p

High nuclear cyclin D1 expression inversely correlates with Ki-67 and EDU proliferation markers post-p14ARF cell cycle –arrest.

<p><b>A.</b> MCF-7 cells were seeded on cover slips for 48h and subsequently treated with 5 mM IPTG for a further 48h. <b>A.</b> Cells were fixed and immunofluorescence stained for cyclin D1, Ki-67, and Hoechst 33342. <i>Column graph inset:</i> Over 400 cells per treatment were counted for nuclear cyclin D1 (red staining) (white bar) and nucleolar Ki-67 (green staining) (black bar) and represented as a percentage of total nuclei (blue staining) (+/− SD). <b>B. EdU incorporation post IPTG (p14ARF) treatment.</b> EdU was added to the medium of live cells 48h post-IPTG treatment and incubated for a further 20h. EdU incorporation was visualised by staining with Alexafluor 488 (green). The nucleus was stained with Hoechst 33342 (blue) and images were taken on a Nikon Ti Eclipse fluorescence microscope (magnification x200). <i>Column graph inset:</i> % cells staining for EdU compared to Hoechst 33342 stained nuclei (+/− IPTG). A minimum of 400 cells was counted for each treatment. These experiments were performed in duplicate on two separate occasions.</p

Abnormal nuclear growth and proliferation is associated with co-expression of cyclin D1 and Ki-67 in the nucleolus of latent p14ARF cell cycle arrested cells.

<p>MCF-7 cells were seeded on cover slips for 48h and subsequently treated with 5 mM IPTG for a further 11 days. <b>A.</b> Cells were fixed and immunofluorescent stained for cyclin D1 (red staining), Ki-67 (green staining), and Hoechst 33342 (blue staining). <b>B.</b> Cells were stained for p14ARF (red staining). Images were taken on a Nikon fluorescence microscope (magnification x200).</p

Escape from p14ARF cell cycle arrest (premature senescence in MCF-7 breast cancer cells): A proposed model.

<p>P14ARF binds hdm2 and sequesters it to the nucleolus. P53 upregulates p21 and p21 dephosphorylates and downregulates Rb. P21 also binds and inactivates cyclin D1/cdk4/6 complex and induces premature senescence. Two avenues are suggested whereby cells may escape from senescence: ER binds p53 and inactivates p14ARF-p53 inhibitory functions <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042246#pone.0042246-Konduri1" target="_blank">[44]</a>; alternatively ER binds to cyclin D1 with a gain of cell proliferative function <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042246#pone.0042246-Zwijsen1" target="_blank">[40]</a>.</p

Cell cycle recurrence is abrogated by anti-estrogen ICI 182780.

<p>Cells were seeded at 200 cells per well and treated with combinations of IPTG, ICI 182780, E2 and vehicle over a period of 4 and 12 weeks and colonies were visualised with methylene blue staining. <b>A.</b> Colonies <u>(+)</u> were counted using a Bio-Rad ChemiDoc XRS + imager in biological triplicate. <b>B.</b> Representative wells showing IPTG and ICI 182780 and vehicle (control) treated colonies at week 4. <b>C.</b> IPTG treated cells at week 12.</p

LE and lipoaspirate tissue samples.

<p>(A) Left arm LE, (B) Left leg LE, (C) LE AT liposuction aspirate and (D) Normal non-LE (cosmetic surgery) AT liposuction aspirate, showing presence of liquid oil (Oil) and solid fat (Fat). Also evident is the aqueous layer of the saline wash within the cosmetic surgery aspirate.</p

Correlation analysis of altered AT fatty acids and phospholipid abundance with LE years.

<p>Correlation analysis of altered AT fatty acids and phospholipid abundance with LE years.</p

Study cohort characteristics.

<p>Study cohort characteristics.</p