Figure 1.
NPM-ALK expression in primary MEFs inhibits cellular proliferation.
(A) Experimental design and reference timeframe. Infection refers to exposure of MEFs to retrovirus, and selection refers to enrichment for transduced cells with 2 µg/ml of puromycin. (B) MEFs infected with retrovirus and selected for expression of the indicated genes were assessed for their ability to proliferate over 6 days via crystal violet assay. Relative cell growth was determined by comparing crystal violet optical density values obtained at 590 nm at each time point with those obtained at day 0. Each time point was conducted in triplicate in at least 3 separate experiments using MEFs from independent embryo preparations. Error bars are standard deviations of the mean.
Figure 2.
NPM-ALK expression in primary MEFs induces a senescence-like arrest.
(A) Representative data of flow cytometric cell-cycle analysis following BrdU incorporation and PI staining. The assay was performed at day 6. Lower-left box identifies cells with 2n DNA content; upper box identifies cells in S phase; Lower-right box identifies cells with 4n DNA content. (B) Graphical representation of cell-cycle analysis (left panel). Data shows the mean of three independent experiments. Error bars are standard deviations of the mean. *denotes significance at p<0.05 using a Student's T-test. Microscopic examination at ×100 magnification showing NPM-ALK cells with 2 nuclei (indicated by arrows) (right panel). (C) Protein lysates prepared from cells transduced with and selected for expression of vectors encoding NPM-ALK (NA) or an empty vector (EV) were examined by Western blot for expression of the indicated proteins with their respective antibodies (pp-Rb indicates phosphorylated Rb). (D) Bright-field images of MEFs (×25 magnification) infected with the indicated retroviruses and assayed for SA-β-Gal activity (indicated by blue coloring) at day 4 (right panel). The bar chart displays quantification of the senescence assay. H-Ras-V12-expressing MEFs were used as a positive control for senescence. At least 200 cells were counted per well (in triplicate in at least 3 separate experiments using MEFs from independent embryo preparations). Error bars are standard deviations of the mean. **denotes significance at p<0.01 using a Student's T-test.
Figure 3.
NPM-ALK-induced senescence is p53-dependent.
Early-passage p53−/− MEFs were retrovirally transduced with empty (EV), NPM-ALK- (NA), or H-Ras-V12 (Ras)-containing vectors and selected in puromycin as detailed in Fig. 1A before use in the following experiments: (A) Relative cell growth was determined by comparing crystal violet optical density values obtained at 590 nm at each time point with those obtained at day 0. (B) SA-β-Gal activity was determined at day 4 post-selection. At least 200 cells were counted per well. (C) Anchorage-independent growth in soft agarose was assessed by foci formation at weeks 2–3. Each experiment in (A)–(C) was conducted in triplicate in at least 2 separate experiments using MEFs from independent embryo preparations. Error bars are standard deviations of the mean. **denotes significance at p<0.01 using a Student's T test.
Figure 4.
NPM-ALK-induced senescence is p53-dependent but is not due to protein stabilization.
(A) Western blot demonstrating lack of p53/p21/p16/p19 up-regulation in response to NPM-ALK expression in MEFs at the indicated time points in comparison to oncogenic H-Ras V12-expressing cells. (B) NPM-ALK-expressing MEFs treated with the MDM2-antagonist Nutlin-3 for the indicated times show stabilization of p53 and transcriptional activity as evidenced by p21 expression, detected by Western blot. (C) CD2/NPM-ALK transgenic mice when back-crossed onto a p53 heterozygous background displayed a significant increase in tumor development suggesting that p53 delays tumor progression.
Figure 5.
Acute mutation of Rb (but not germline loss of p16) permits escape from NPM-ALK-induced senescence yet the cells remain sensitive to p53-dependent barriers to transformation.
(A) Early-passage p16−/− MEFs were retrovirally transduced with empty (EV), NPM-ALK- (NA), or H-Ras-V12 (Ras)-containing vectors and selected in puromycin as detailed in Fig. 1A before use in a crystal violet assay to assess proliferative capacity. Relative cell growth was determined by comparing crystal violet optical density values obtained at 590 nm at each time point with those obtained at day 0. Each time point was conducted in triplicate in at least 3 separate experiments using MEFs from independent embryo preparations. Error bars are standard deviations of the mean. (B) Floxed cRblox/lox (Rb-null) or control cRblox/lox (Rb-WT) MEFs were transduced by retrovirus with empty (EV) or NPM-ALK- (NA) containing vectors as indicated and used in 3T3 assays to assess long-term proliferative potential. The data shown are representative of two independent cRblox/lox MEF populations derived from two different embryos. (C) Western blot analysis showing levels of the indicated proteins in empty-vector and NPM-ALK-expressing conditional Rb-deficient (Rb-null) MEFs at late passage (cells post-senescent at passages 18 and 15 are shown from 2 independent MEF preparation experiments). Horizontal lines on the Western blot indicate repositioning of the gel lanes. (D) Top panel: images of the soft agarose assay (×25 magnification) demonstrating that whilst NPM-ALK-expressing cells acutely mutated for Rb have escaped senescence they do not grow in an anchorage-independent manner, and therefore are not transformed (for quantification of data see table 1). Inactivation of p53 by GSE56 expression in these cells results in transformation (lower right box). Bottom panel: Western blot showing NPM-ALK and p53 expression in the different cells used in the soft agarose assay. Experiments were performed twice with two independent MEF populations. (E) p53 is induced following NPM-ALK expression in cells acutely mutated for Rb. Lysates were prepared from MEFs of the indicated genotypes and the levels of Rb, p53, p19ARF and NPM-ALK determined by Western blot. “Rb-null” denotes MEFs in which the Rb gene is acutely mutated by addition of Adenovirus expressing Cre-recombinase, and “Rb-WT” denotes MEFs infected with a control Cre-recombinase-deficient Adenovirus.
Table 1.
Loss of p53 function is required for anchorage-independent growth of NPM-ALK-expressing conditional Rb-null post-senescent MEFs.
Figure 6.
NPM-ALK paradoxically induces transcription and degradation of p53 to generate an equilibrium whereby the dominating residual levels of p53 are sufficient to induce senescence.
We propose that NPM-ALK induces transcription of p53 whilst also stabilizing MDM2 and Rb with the effect of attenuating p53 activity. However, equilibrium is reached whereby residual p53 protein is sufficient to set in place a senescence pathway.