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. 2005 Jan;25(1):422-31.
doi: 10.1128/MCB.25.1.422-431.2005.

Growth inhibition by the tumor suppressor p33ING1 in immortalized and primary cells: involvement of two silencing domains and effect of Ras

Frauke Goeman  1 , Dorit Thormeyer, Maria Abad, Manuel Serrano, Oliver Schmidt, Ignacio Palmero, Aria Baniahmad
Affiliations

Growth inhibition by the tumor suppressor p33ING1 in immortalized and primary cells: involvement of two silencing domains and effect of Ras

Frauke Goeman et al. Mol Cell Biol. 2005 Jan.

Abstract

ING1 was identified as an inhibitor of growth and has been described as a tumor suppressor. Furthermore, the expression of ING1 is induced in senescent cells and antisense ING1 extends the proliferative life span of primary human fibroblasts. Cooperation of p33ING1 with p53 has been suggested to be an important function of ING1 in cell cycle control. Intriguingly, it has been shown that p33ING1 is associated with histone acetylation as well as with histone deacetylation function. Here we show that p33ING1 is a potent transcriptional silencer in various cell types. However, the silencing function is independent of the presence of p53. By use of deletion mutants two potent autonomous and transferable silencing domains were identified, but no evidence of an activation domain was found. The amino (N)-terminal silencing domain is sensitive to the histone deacetylase inhibitor trichostatin A (TSA) whereas the carboxy-terminal silencing function is resistant to TSA, suggesting that p33ING1 confers gene silencing through both HDAC-dependent and -independent mechanisms. Interestingly, the presence of oncogenic Ras, which is able to induce premature senescence, increases the p33ING1-mediated silencing function. Moreover, ING1-mediated silencing was reduced by coexpressing dominant-negative Ras or by treatment with the mitogen-activated protein kinase inhibitor PD98059 but not by treatment with SB203580, an inhibitor of the p38 pathway. In addition, we show that both silencing domains of ING1 are involved in cell cycle control, as measured by inhibition of colony formation of immortalized cells and by thymidine incorporation of primary human diploid fibroblasts (HDF). Interestingly, p33ING1 expression induces features of cellular senescence in HDFs.

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Figures

FIG. 1.
FIG. 1.
The p33ING1 tumor suppressor protein harbors a potent transferable silencing domain functional in various cell types. (A) Schematic view of p33ING1 and its splice variant p24ING1 and homologies to other members of the ING1 protein family, which are encoded by different genes. The ING1 protein family shares a tripartite structure: a conserved N terminus, a nonconserved central part, and a highly conserved carboxy terminus. The percentages of identical amino acids in relation to p33ING1 are indicated. The positions of the previously identified mutations of p33ING1 are highlighted (white lines). PHD, plant homeodomain. (B) Various cell lines were cotransfected with expression vectors coding for the Gal4 DNA binding domain Gal94 (amino acids 1 to 94) or the Gal-p33ING1 fusion together with the reporter 17mer6x-tkCAT. The mean of the values obtained with Gal94 was set arbitrarily at 1, and the repression by the p33ING1 fusion is indicated. The data were normalized to the values obtained with the cotransfected β-Gal expression vector (pCMV-lacZ). The error bars represent deviations of the means from two independent transfections. Please note the different scaling parameters for individual graphs.
FIG. 2.
FIG. 2.
Silencing function of p33ING1 is independent of the tumor suppressor p53. (A) The human lung carcinoma cell line H1299, lacking functional p53, was cotransfected with either Gal-1-94 or Gal-p33ING1 together with the reporter 17mer6x-tkCAT. The mean of the values obtained with Gal94 was set arbitrarily at 1, and the severalfold repression obtained by the p33ING1 fusion is indicated. Black bars indicate values obtained with cotransfected p53 expression vector (100 ng), and gray-shaded bars indicate the values obtained with the empty control vector. The transfection efficiency was normalized with lacZ values. (B) As a control for p53 function in H1299 cells, the human telomerase promoter (Δ1009 TERT-Luc) was used. The mean value obtained in the absence of p53 (empty vector) was set arbitrarily at 1 (gray bar). Cotransfection of 50 and 500 ng of the p53 expression vector led to repression of the TERT promoter (black bars). The error bars represent deviations of the means from two independent transfections.
FIG. 3.
FIG. 3.
Delineation of the silencing domains of p33ING1. The transcriptional effect of the indicated gal-p33ING1 deletion mutants on the activity of the reporter, 17mer6x-tkCAT, was analyzed in CV1, HEK 293T, and H1299 cells. The values obtained with Gal alone were set arbitrarily at 1. Values were normalized for transfection efficiency with lacZ values. The error bars represent the variations from the means of three independent transfections. The numbers indicate the positions of amino acids of the p33ING1 deletions. The gray underlying shadowed boxes indicate the two strong silencing domains in the N and C terminus of p33ING1. The error bars represent deviations of the means from two independent transfections.
FIG. 4.
FIG. 4.
Differential effects of TSA on the C- and N-terminal p33ING1 silencing domains. The HDAC inhibitor TSA (100 ng/ml) was used to test for HDAC-sensitive or -resistant silencing by treatment of CV1 cells transfected with full-length p33ING1 or deletions lacking the N-terminal or C-terminal silencing domain of p33ING1. Values obtained with Gal94 in the absence of TSA were set arbitrarily at 1. As a positive control for TSA-sensitive silencing, Gal-NCoR was used. The error bars represent deviations of the means from two independent transfections.
FIG. 5.
FIG. 5.
The Ras signal transduction pathway enhances p33ING1-mediated gene silencing. (A) The effect of dominant active Ras (RasV12), dominant-negative Ras (RasN17), or constitutive active Raf (Raf BXB) on the Gal-p33ING1 full-length fusion in CV1 cells was analyzed. Expression vectors were cotransfected with the reporter UAS4x-tkLuc, and each experimental setup contained four independent transfections. The values obtained with Gal alone were set arbitrarily at 1. Values were normalized for transfection efficiency with lacZ. C, empty expression vector. (B) The inhibitor of MAPK (MEK) signaling PD98059 (PD) (10 μM), the p38 kinase inhibitor SB203580 (SB) (25 μM), and DMSO (vehicle) as a control were tested for their influence on p33ING1-mediated silencing in a setup similar to that described for panel A. The values obtained with Gal alone were set arbitrarily at 1. Values were normalized for transfection efficiency with lacZ. Only PD98059 inhibits p33ING1-mediated silencing.
FIG. 6.
FIG. 6.
The highly conserved ING1 C terminus is involved in cell growth regulation. A colony formation assay was performed using immortalized NIH 3T3 cells transfected with selectable expression vectors for full-length p33ING1 and the indicated ING1 deletions. The cell colonies were stained with crystal violet after selection with antibiotics for 2 weeks (left panel); the colony numbers were then counted and plotted in a graph (right panel). Each experiment set contained triplicates for the procedures performed for each of the constructs, which were repeated three times.
FIG. 7.
FIG. 7.
ING1 induces morphological changes and growth arrest of primary human diploid fibroblasts. The human primary diploid fibroblasts expressing an ecotropic receptor were retrovirally transformed with selectable expression plasmids for the oncogenic Ras, RasV12, full-length p33ING1, and the indicated ING1 deletions. (A) Thymidine incorporation as an indicator of DNA synthesis. Triplicate experiments were performed, and the values obtained with the empty vector control were set arbitrarily at 100%. (B) Phase-contrast pictures of transduced human diploid fibroblasts with the indicated expression vectors. (C) Expression of SA-β-Gal. A total of 200 human primary diploid fibroblasts were counted each time; the percentages of cells expressing SA-β-Gal are shown.
FIG. 7.
FIG. 7.
ING1 induces morphological changes and growth arrest of primary human diploid fibroblasts. The human primary diploid fibroblasts expressing an ecotropic receptor were retrovirally transformed with selectable expression plasmids for the oncogenic Ras, RasV12, full-length p33ING1, and the indicated ING1 deletions. (A) Thymidine incorporation as an indicator of DNA synthesis. Triplicate experiments were performed, and the values obtained with the empty vector control were set arbitrarily at 100%. (B) Phase-contrast pictures of transduced human diploid fibroblasts with the indicated expression vectors. (C) Expression of SA-β-Gal. A total of 200 human primary diploid fibroblasts were counted each time; the percentages of cells expressing SA-β-Gal are shown.
FIG. 7.
FIG. 7.
ING1 induces morphological changes and growth arrest of primary human diploid fibroblasts. The human primary diploid fibroblasts expressing an ecotropic receptor were retrovirally transformed with selectable expression plasmids for the oncogenic Ras, RasV12, full-length p33ING1, and the indicated ING1 deletions. (A) Thymidine incorporation as an indicator of DNA synthesis. Triplicate experiments were performed, and the values obtained with the empty vector control were set arbitrarily at 100%. (B) Phase-contrast pictures of transduced human diploid fibroblasts with the indicated expression vectors. (C) Expression of SA-β-Gal. A total of 200 human primary diploid fibroblasts were counted each time; the percentages of cells expressing SA-β-Gal are shown.
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