DNA damage-inducible gene p33ING2 negatively regulates cell proliferation through acetylation of p53

Abstract

The p33ING1 protein is a regulator of cell cycle, senescence, and apoptosis. Three alternatively spliced transcripts of p33ING1 encode p47ING1a, p33ING1b, and p24ING1c. We cloned an additional ING family member, p33ING2/ING1L. Unlike p33ING1b, p33ING2 is induced by the DNA-damaging agents etoposide and neocarzinostatin. p33ING1b and p33ING2 negatively regulate cell growth and survival in a p53-dependent manner through induction of G(1)-phase cell-cycle arrest and apoptosis. p33ING2 strongly enhances the transcriptional-transactivation activity of p53. Furthermore, p33ING2 expression increases the acetylation of p53 at Lys-382. Taken together, p33ING2 is a DNA damage-inducible gene that negatively regulates cell proliferation through activation of p53 by enhancing its acetylation.

Figure 1

p33ING1b and p33ING2 protein expression. Expression of p33ING1b, p33ING2, and actin was analyzed in 12 human cell lines by Western blotting. The steady-state levels of p33ING1b and p33ING2 were quantified by densitometry and normalized with actin.

Figure 2

p53, p33ING1b, and p33ING2 protein expression, and p53 acetylation after DNA damage (A–E). After cells were treated with 10 Gy of γ-irradiation (IR), 0.2 μg/ml doxorubicin (DOX), 30 μg/ml etoposide (ETO), 0.3 μg/ml neocarzinostatin (NCS), 0.03 units/ml bleomycin (BLEO), or 2 μg/ml cis-platinum (CDDP), protein expression of p53, p33ING1b, p33ING2, and actin and p53 acetylation at Lys-382 at each time point were analyzed by Western blotting. (F) The steady-state level of each protein was quantified by densitometry and normalized with actin. p53, p33ING1b, p33ING2, and p53 acetylation at Lys-382 after exposure to ETO.

Figure 3

Negative regulation of cell proliferation by p33ING1b or p33ING2. RKO or RKO E6 cells were transfected with empty control, p33ING1b, or p33ING2 expression vector. (A) Colony-formation assay. After 2 weeks of G418 selection, colonies were fixed, stained with crystal violet, and counted. Data represent the means ± SD. Statistical analysis was carried out with the Student's t test. (B) Induction of apoptosis by p33ING1b or p33ING2 was analyzed. Data represent the means ± SD of three independent experiments. Statistical analysis was carried out with the Student's t test. (C) Cell-cycle regulation by p33ING1b or p33ING2 was analyzed by flow cytometry. Cell-cycle profiles (G₀/G₁, S, and G₂/M) were calculated with the modfit program.

Figure 4

Sequence-specific transcriptional-transactivation activity of p53. Promoter activities of p21/waf1 and bax were detected after cotransfection of p53-responsive reporter constructs with empty control, p33ING1b, or p33ING2 expression vectors in RKO or RKO E6 cells. The luciferase activities from the reporter constructs were measured with a luminometer. Relative luciferase activities (RLAs) were calculated by dividing the luciferase activity by the protein concentration. Results represent the means ± SD of six independent experiments. Differences in the relative luciferase activities were analyzed by using the Student's t test.

Figure 5

Interaction of p53 with p33ING1b- and p33ING2-mediated acetylation of p53. Immunoprecipitation (IP)-Western analyses were performed with RKO or OsA-CL cell extracts after transfection with empty control, p33ING1b, p33ING2, or anti-p33ING2 expression vector. (A) Expression of p53, p33ING1b, p33ING2, and actin in RKO cells after transfection was detected by Western blot analysis. (B) Anti-p53 (Ab-6 and Pab 240), anti-p33ING1b (Ping1), normal mouse IgG (negative control), or normal rabbit IgG (negative control) were used for immunoprecipitation to analyze the interaction of p53 with p33ING1b in RKO cells. (C) Acetylation of p53 at Lys-382 was detected in anti-p53 (Ab-6 and Pab 240) immunoprecipitates from RKO cells. (D) Phosphorylation of p53 at either Ser-15 or Ser-392 was investigated in RKO cells by Western blot analysis. C3ABR cells were treated with doxorubicin for 8 hr and were used as positive controls for acetylated and phosphorylated p53. (E) Acetylation of p53 at Lys-382 in OsA-CL cells treated for 8 hr with ETO 24 hr after transfection with empty control or anti-p33ING2 expression vector.