3621

ING1

inhibitor of growth family, member 1;ING1;inhibitor of growth family, member 1

growth inhibitor ING1|growth inhibitory protein ING1|inhibitor of growth protein 1|tumor suppressor ING1

13q34

protein-coding

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.

OBJECTIVES: To investigate the pharmacological effects of azidothymidine (AZT) on p33ING1b expression, senescence and apoptosis of TJ905 glioblastoma cells. METHODS: TJ905 cells were treated with AZT at a serial concentrations of 50, 100 and 200 micromol/L. Semi-quantitative RT-PCR and cytochemical staining of senescence related-galactosidase (sbeta-Gal) were used to evaluate the expression of p33ING1b mRNA and to label the senescent cells at the 1st, 3rd and 6th generations, respectively. In situ cell death detection and single cell gel electrophoresis were used to detect the apoptosis at the 3rd and 6th generations. RESULTS: AZT induced the expression of p33ING1b mRNA and senescence of the tumor cells of the 1st generation in a dosage and time dependent manner. At the 6th generation, the relative amount of p33ING1b RT-PCR product (1.44+/-0.23) and sbeta-Gal labeling index of 200 micromol/L group (45.62+/-6.74) were significantly higher than those of the 1st (0.95+/-0.13 and 7.82+/-2.40) and the 3rd generation cells (1.35+/-0.23, 26.27+/-7.17) of the same group, and cells of the same generation in the 50 micromol/L (0.85+/-0.24, 27.37+/-6.41) and 100 micromol/L groups (1.23+/-0.34, 35.49+/-5.12, P<0.01). There was a significant positive correlation between the p33ING1b mRNA expression and the labeling index of sbeta-Gal. Pro-apoptotic effects of AZT became obvious at the 6th generation. CONCLUSION: AZT upregulates the expression of p33ING1b, a possible mechanism in regulating senescence and apoptosis of the TJ905 cells.

ING1 protein is a tumor suppressor which plays significant roles in multiple cellular activities. p47(ING1a) and p33(ING1b) are major splice isoforms of ING1 and their roles in senescence need further investigations. Here we studied the functions of ING1 isoforms in cellular senescence and gene regulation, with focus on p16(INK4a). We observe that p33(ING1b) protein is the major ING1 isoform expressed in 2BS human diploid fibroblasts. Overexpression of p33(ING1b) induces cellular senescence and upregulates p16(INK4a) expression in 2BS fibroblasts. p33(ING1b) upregulates p16(INK4a) transcription. p33(ING1b) and p300 bind to the p16(INK4a) promoter. p300/CBP-specific inhibitor curcumin can reverse the induction of p16(INK4a) by p33(ING1b). These results help to better understand the function of ING1.