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1.

Figure 7. From: PML is induced by oncogenic ras and promotes premature senescence.

Colocalization of PML, Rb, and p53 by laser scanning confocal microscopy. (A) Representative images of IMR90 cells containing an empty vector (V) or oncogenic ras (R) costained with anti-pRb and anti-PML antibodies. (B) Representative images of IMR90 cells containing an empty vector (V) or oncogenic ras (R) costained with anti-p53 and anti-PML antibodies. (C) Representative images of cells expressing a p53-green fluorescent protein (GFP) fusion protein and either a control vector (V), oncogenic ras (R), or PML (P). In this series, p53 was localized by direct GFP fluorescence, whereas PML was localized using an anti-PML antibody.

Gerardo Ferbeyre, et al. Genes Dev. 2000 August 15;14(16):2015-2027.
2.
Figure 5

Figure 5. From: PML is induced by oncogenic ras and promotes premature senescence.

PML induces premature senescence. (A) IMR90 cell populations containing an empty vector (V), oncogenic ras (R) or PML were stained for SA-β-gal 6 days postselection. IMR90 cells that were serially passaged until senescent (S) are shown for comparison. Representative photomicrographs are shown; senescent-cells display a cytoplasmic blue stain. (B) Quantitation of data in A, showing the percentage of SA-β-gal positive cells. The data represent the mean ± standard deviation of three independent counts of 200 cells. (C) Expression of PAI-1 in the indicated populations was measured by Northern blot.

Gerardo Ferbeyre, et al. Genes Dev. 2000 August 15;14(16):2015-2027.
3.
Figure 8

Figure 8. From: PML is induced by oncogenic ras and promotes premature senescence.

A tumor suppressor network is activated by oncogenic ras and inhibited by E1A in human diploid fibroblasts. Expression of oncogenic ras in human diploid fibroblasts induces first uncontrolled proliferation and then a permanent cell-cycle arrest with characteristics of cellular senescence. The process involves the mitogen-associated protein (MAP) kinase pathway () and the tumor suppressors p53, p16, Rb () and PML (this paper). PML modulates both the Rb and p53 pathways but may have additional activities. Lesions that alter individual tumor suppressors in isolation do not override ras-induced arrest (; ). However, E1A disables multiple tumor-suppressor pathways and, hence, overrides ras-induced arrest (see bars).

Gerardo Ferbeyre, et al. Genes Dev. 2000 August 15;14(16):2015-2027.
4.
Figure 4

Figure 4. From: PML is induced by oncogenic ras and promotes premature senescence.

Characteristics of PML-induced arrest. (A) Growth curves of IMR90 cells containing empty vector (ς), oncogenic ras (λ) or PML (ν). Each value was determined in triplicate and normalized to the cell number at day 0, which corresponded to the time in which the cells finished drug selection. (B) Cell-cycle analysis of populations containing an empty vector, oncogenic ras or PML. The cells were pulsed with BrdU for 4 hours and analyzed for BrdU uptake and DNA content by two-color flow cytometry using a fluorescein isothiocyanate (FITC)-conjugated anti-BrdU antibody and propidium iodide (see Materials and Methods). The percentage of BrdU incorporation is indicated at the top of each panel.

Gerardo Ferbeyre, et al. Genes Dev. 2000 August 15;14(16):2015-2027.
5.
Figure 6

Figure 6. From: PML is induced by oncogenic ras and promotes premature senescence.

PML engages the Rb and p53 pathways. (A) The expression of Rb, cyclin A, p16, and p53 was measured by immunoblotting of lysates from IMR90 cells containing an empty vector (V), oncogenic ras (R) or PML (P). (B) Expression of p53 targets p21 and mdm2 by Northern blot analysis of total RNA extracted from the cells indicated above. (C) p53 phosphorylation on serine 15 was detected by immunoblotting using antibodies that specifically bind PS-15 p53. Lysates were prepared from cells infected with a empty vector (V), oncogenic ras (R), wild-type human p53 (p53), PML (P), or both the p53 and PML expressing viruses (p53 +P). The expression of total p53 was measured using a polyclonal antibody that recognizes all p53. The expression of the p53-target p21 is shown for comparison. Lysates were prepared from cells containing an empty vector (V), oncogenic ras (R), p53, PML (P) and a combination of p53 and PML (p53 +P). (D) The impact of E1A on PS-15 p53 was determined as in B, in which (ER) indicates cells co-expressing E1A and oncogenic ras.

Gerardo Ferbeyre, et al. Genes Dev. 2000 August 15;14(16):2015-2027.
6.
Figure 1

Figure 1. From: PML is induced by oncogenic ras and promotes premature senescence.

Oncogenic ras induces PML. (A) 33P-labeled cDNA probes were generated from quiescent (top) and ras-arrested (bottom) IMR90 fibroblasts and used to hybridize to GDA arrays. The portion of each array that contains the PML cDNA (n-5, see arrows) is shown; each cDNA is arrayed in duplicate and in different orientations (see www.genomesystems.com). Note that the cDNAs located at p-1 and 1–1 are controls that show equal intensity between the filters, whereas several other cDNAs in this section displayed up- or down-regulation in ras-arrested cells. (B) Northern blot analysis of PML and the senescence-related genes PAI-1, stromelysin, and IL-1β. IMR90 cells were infected with a control vector (V), oncogenic ras (R), or coinfected with E1A and oncogenic ras (ER). Alternatively, the cells were serially passaged until they had become senescent (S). Three major transcripts were visualized for PML (). The smaller of the two PAI-1 transcripts is the one whose up-regulation correlates better with senescence ().

Gerardo Ferbeyre, et al. Genes Dev. 2000 August 15;14(16):2015-2027.
7.
Figure 2

Figure 2. From: PML is induced by oncogenic ras and promotes premature senescence.

PODs accumulate during premature senescence. PML and Sp100 localization in IMR90 cells was determined by indirect immunofluorescence using the PGM3 monoclonal antibody and the rabbit Anti-Sp100 polyclonal antibody AB1380. To facilitate comparison, photomicrographs of each cell were prepared using identical microscope settings and exposure times. In all instances, similar results were obtained with a polyclonal antibody directed to a different epitope of human PML (see Materials and Methods). (A) Confocal images of costaining for PML and Sp100 in cells expressing an empty vector (V), oncogenic ras (R) and senescent cells (S). (B) PML staining in a representative series of cells expressing an empty vector (V) and oncogenic ras (R). (C) PML staining in exponentially growing subconfluent (SC) cells was compared to cells arrested by confluence (C), low serum (LS), or enforced p53 expression. (D) PML staining in cells expressing E1A or coexpressing E1A and oncogenic ras. (E) PML staining of ras-expressing cells fixed 2, 4, 6, and 10 days after selection for cells expressing oncogenic ras. In panels B–D, the percentage of SA-β-gal positive cells in each population is indicated below each picture.

Gerardo Ferbeyre, et al. Genes Dev. 2000 August 15;14(16):2015-2027.
8.

Figure 3. From: PML is induced by oncogenic ras and promotes premature senescence.

Impact of enforced PML expression on IMR90 cells. (A) IMR90 cells or IMR90 cells expressing E1A were infected with a control vector (V) or a virus expressing PML. PML staining and POD structure were visualized by indirect immunofluorescence using 4′6-diamidino-2-phenylindole (DAPI) staining to highlight the nucleus. Representative samples are shown. The arrow in the bottom right panel (E1A) points to a cytoplasmic PML conglomerate. (B) Cell morphology and in situ bromodeoxyuridine (BrdU) incorporation of control IMR90 cells or E1A-expressing IMR90 (E1A) cells infected with an empty vector (V), or viruses expressing oncogenic ras (R) or PML (P). The cells were pulsed with 10 μm BrdU for 2 hours a day 4 postselection, and BrdU incorporation was visualized using immunohistochemistry. (C) Quantitation of the results presented in B, showing the percentage of BrdU-positive nuclei. The average and standard deviation of three independent counts of 200 cells are shown. The abbreviations are as follows: (V) vector; (R) oncogenic ras; (P) PML; (E) E1A. (D) 3H-thymidine incorporation in cells containing a vector control (V), PML, oncogenic ras, or two PML mutants, PML Nls- and PML prb-. All cell populations were pulsed with 3H-thymidine for 24 hours, beginning 3 days postselection and the amount of incorporation was measured as described in Materials and Methods. Each point represents the mean ± standard deviation of data obtained in three independent experiments.

Gerardo Ferbeyre, et al. Genes Dev. 2000 August 15;14(16):2015-2027.

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