Growth Inhibition by the Tumor Suppressor p33ING1 in Immortalized and Primary Cells: Involvement of Two Silencing Domains and Effect of Ras

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.

ACKNOWLEDGMENTS

We greatly thank J. M. Freije and C. Lopez-Otin for providing the p33ING1 cDNA, M. Dobbelstein for the p53 expression vector and H1299 cells, S. Bacchetti and A. Farsetti for providing the human telomerase promoter (TERT) construct, P. Crespo for Ras and Raf expression vectors, M. Rosenfeld for pCMX-gal-NCoR, G. Piaggio and K. Katula for the cyclin B1 reporter, and K. W. Scotto for the MDR1 reporter plasmid.

Part of this work was supported by the Graduiertenkolleg 370 (F.G.). This work has been supported by grants from the Spanish Ministry of Education (SAF03-00801) and the Cooperative Cancer Network of the Spanish Ministry of Health.