p16INK4A Positively Regulates Cyclin D1 and E2F1 through Negative Control of AUF1

/pRB/E2F pathway, a key regulator of the critical G1 to S phase transition of the cell cycle, is universally disrupted in human cancer. However, the precise function of the different members of this pathway and their functional interplay are still not well defined. -dependent manner, and several of these genes are also members of the AUF1 and E2F1 regulons. We also present evidence that E2F1 mediates p16-dependent regulation of several pro- and anti-apoptotic proteins, and the consequent induction of spontaneous as well as doxorubicin-induced apoptosis. is also a modulator of transcription and apoptosis through controlling the expression of two major transcription regulators, AUF1 and E2F1

Methylation of BRCA1 and MGMT genes in white blood cells are transmitted from mothers to daughters

Abstract Background Constitutive methylation of tumor suppressor genes are associated with increased cancer risk. However, to date, the question of epimutational transmission of these genes remains unresolved. Here, we studied the potential transmission of BRCA1 and MGMT promoter methylations in mother-newborn pairs. Methods A total of 1014 female subjects (cancer-free women, n = 268; delivering women, n = 295; newborn females, n = 302; breast cancer patients, n = 67; ovarian cancer patients, n = 82) were screened for methylation status in white blood cells (WBC) using methylation-specific PCR and bisulfite pyrosequencing assays. In addition, BRCA1 gene expression levels were analyzed by quantitative real-time PCR. Results We found similar methylation frequencies in newborn and adults for both BRCA1 (9.9 and 9.3%) and MGMT (12.3 and 13.1%). Of the 290 mother-newborn pairs analyzed for promoter methylation, 20 mothers were found to be positive for BRCA1 and 29 for MGMT. Four mother-newborn pairs were positive for methylated BRCA1 (20%) and nine pairs were positive for methylated MGMT (31%). Intriguingly, the delivering women had 26% lower BRCA1 and MGMT methylation frequencies than those of the cancer-free female subjects. BRCA1 was downregulated in both cancer-free woman carriers and breast cancer patients but not in newborn carriers. There was a statistically significant association between the MGMT promoter methylation and late-onset breast cancers. Conclusions Our study demonstrates that BRCA1and MGMT epimutations are present from the early life of the carriers. We show the transmission of BRCA1 and MGMT epimutations from mother to daughter. Our data also point at the possible demethylation of BRCA1and MGMT during pregnancy

Schematic representation of the p16-related regulation of AUF1,

<p>E2F1 and CyclinD1. See text for details.</p

p16 modulates apoptosis through E2F1.

<p>(A) Western blots showing the expression of the indicated pro-and anti-apoptotic proteins in U2OS and EHI. (B) Western blots showing the expression of the indicated proteins in U2OS, U2OS cells stably transfected with plasmids encoding either scrambled sequence (ctl) or E2F1 (+). The numbers below the bands indicate the corresponding expression levels. (C) U2OS, EH1 and E2F1-expressing U2OS cells were either mock-treated or challenged with doxorubicin (2 µM) and then re-incubated for 72 hrs. Cells were then divided into two groups; one was used to analyze cell death by annexinV/PI flow cytometry. The numbers in the charts indicate the proportions of early and late apoptosis. (D) Histogram showing the proportions of apoptosis (early+late) induced by different doses of doxorubicin. The error bars represent standard deviation of three different experiments. (E) The second group of cells was used to assess the level of the indicated proteins by immunoblotting. (F) Graph showing the Bax/Bcl-2 ratio in the indicated cells after treatment with doxorubicin. Error bars represent standard deviation of at least three different experiments.</p

p16 modulates E2F1 and cyclin D1 protein and mRNA levels in human and mouse cells.

<p>Whole cell extracts and total RNA were prepared from different human and mouse cell lines. (A–C) Western blots using the indicated antibodies. The histogram shows the expression levels of the indicated proteins. EH2 cells were treated with IPTG at 1 mM (D) Upper panel, ethidium bromide stained agarose gels showing RT-PCR products of the indicated genes. The numbers below the bands indicate the corresponding expression levels relative to β-actin. These experiments were repeated several times and representative ones are shown. The histogram shows data of real time RT-PCR of the indicated genes. Error bars indicate standard errors of 3 different experiments.</p

List of genes under the control of both p16 and E2F1.

<p>List of genes under the control of both p16 and E2F1.</p

List of genes under the control of both p16 and AUF1.

<p>*: The variation in the expression of these genes was less than 2 fold in microarray analysis data. However, the variation was significant when RT-PCR was used for assessment, as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021111#pone-0021111-g001" target="_blank">figure 1D</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021111#pone-0021111-g007" target="_blank">figure 7</a>.</p

Involvement of ARE in the E2F1 3′UTR and response to AUF1 and p16.

<p>(A) Schematic diagram of the E2F1 3′UTR, ARE region sequences, and locations. (B) The E2F1 3′UTR sequences in different species (C) Sequences from the E2F1 3′UTR (<i>ARE regions 1 to 3</i>), IL-8 3′UTR (<i>ARE control</i>), and a control that lacks ARE were inserted in BamHI/XbaI sites in EGFP expression vector as shown. The Huh7 cell line (2.10⁴ cells per well) in 96-well black clear-bottom microplates were transfected with the different 3′UTR constructs. The reporter activity was assessed after 24 hr using BD bio-imaging apparatus and software. The non-ARE 3′UTR was used as control and its fluorescence activity was taken as 100%. Data are presented as Mean±SEM (n = 4) of % of the control. *** denote <i>p</i> values of <0.005 (student t- test) when compared to non-ARE control. (D) Huh7 cells (left panel) or U2OS (right panel) in 96-well microplates were co-transfected with siRNA against AUF1 or scrambled control (50 ng per well) and reporter constructs (25 ng per well) as indicated. Reporter activity was assessed at 48 hr post-transfection. Data (Mean±SEM, n = 4) were presented as % increase in reporter fluorescence due to AUF1 silencing when compared to the fluorescence in control-siRNA-treated cells. * and *** denote <i>p</i> <0.05 and <0.005, respectively (student t-test) when compared to non-ARE control. (E) U2OS and EH1 cells (2.10⁴ cells per well) were seeded in 96-well black clear-bottom microplates and then transfected with the different 3′UTR constructs. Reporter activity was assessed as described in (D). ANOVA was performed to compare between U2OS and EH1 data groups.</p