p53DINP1, a p53-Inducible Gene, Regulates p53-Dependent Apoptosis Shu Okamura, Hirofumi Arakawa, Tomoaki Tanaka, Hiroshi Nakanishi, Ching Ching Ng, Yoichi Taya, Morito Monden, Yusuke Nakamura  Molecular Cell  Volume 8, Issue 1, Pages 85-94 (July 2001) DOI: 10.1016/S1097-2765(01)00284-2

Figure 1 Identification of the p53DINP1 Gene (A) Expressions of p53DINP1 and p21WAF1 in the U373MG cell line infected with either Ad-p53 or Ad-LacZ, as detected by Northern blotting. As a quantity control, expression of the β-actin gene was examined in the same cells. Radioactivity was measured with a BAS 1000 Bioimage analyzer (Fujix). (B) Schematic description of the two alternatively spliced transcripts. “M” and asterisks denote the first methionine and the stop codon of each transcript, respectively. Hatching indicates coding regions Molecular Cell 2001 8, 85-94DOI: (10.1016/S1097-2765(01)00284-2)

Figure 2 p53DINP1 Is a Direct-Target Gene of p53 (A) Genomic structure of the p53DINP1 gene. Black boxes indicate locations and relative sizes of the four exons. The location of a potential p53 binding sequence (BS) is indicated in intron 2, and the consensus p53 binding sequence (p53 CBS) is shown above; R, purine; Y, pyrimidine; and W, A, or T. (B) EMSA, anti-p53 antibodies p53Ab1 (Pab421), and p53Ab2 (Pab1801) were present in the designated lanes. Interaction between p53 protein and DNA was inhibited by unlabeled oligonucleotides corresponding to the binding site of the p53DINP1 gene (“self”) but not by nonspecific oligonucleotides (TL). PC-p53BS, the p53 binding sequence within the p21WAF1 gene, served as a positive control. (C) Luciferase assay. Three plasmid constructs containing one copy of 20 bp possible p53 binding sequence (BS) (X1), two copies of BS (X2), or a 477 bp DNA fragment corresponding to intron 2 of the p53DINP1 gene including BS (intron 2-wt) were cloned into luciferase reporter vector containing SV40 minimal promoter. Intron 2-mt reporter vector contains a point mutation which substitutes the fourth nucleotide “C” of BS to “T.” H1299 cells were cotransfected with either of these constructs, pRL-TK vector, and either of wild-type p53 or mutant p53 expression constructs, as well as control reporter vector Molecular Cell 2001 8, 85-94DOI: (10.1016/S1097-2765(01)00284-2)

Figure 3 Expression of p53DINP1 (A) Nothern blotting to detect the expression of mouse p53DINP1 mRNA in response to γ irradiation in p53+/+ and p53−/− MEF. As a quantity control, expression of mouse gapdh gene was examined in the same cells. Radioactivity was measured with a BAS 1000 Bioimage analyzer (Fujix). (B) Western blotting to detect the expression of p53DINP1, p53, and p21WAF1 proteins in MCF7 (p53+/+) cells damaged by γ irradiation, exposure to adriamycin, or UV irradiation. Proteins were collected from cells incubated for different periods of time (0, 0.5, 1, 2, 4, 8, 12, 16, 24, and 48 hr) after exposure to each stress. As a quantity control, expression of actin protein was examined in the same cells. (C) Immunocytochemical staining of p53DINP1 protein in MCF7 cells. Parental cells were stained with DAPI (left), anti-p53DINP1 antibody (center), or both (right) Molecular Cell 2001 8, 85-94DOI: (10.1016/S1097-2765(01)00284-2)

Figure 4 p53DINP1 Is Involved in DSBs-Induced Apoptosis through the Regulation of p53-Ser46 Phosphorylation and p53AIP1 Expression (A) Enhancement in cell survival after DNA damage induced by γ irradiation when expression of p53DINP1 was suppressed by AS2. The average score of the data in three independent experiments is indicated with an error bar. (B) Decrease in cell death in response to γ radiation when expression of p53DINP1 was suppressed by AS2. The experiments were repeated at least three times, and the representative data are shown. (C) Phosphorylation of p53 and proteins of several p53 target genes in MCF7 cells treated with AS2 or SE2, 0, 12, 24, and 48 hr after DNA damage induced by γ irradiation and UV irradiation, detected by Western blotting. As a quantity control, expression of β-actin was examined in the same cells Molecular Cell 2001 8, 85-94DOI: (10.1016/S1097-2765(01)00284-2)

Figure 5 Enhancement of p53-Ser46 Phosphorylation, p53AIP1Expression, and Apoptosis by Cooverexpression of p53DINP1 with p53 (A) Expression of several p53 target mRNAs in MCF7 cells, 0, 12, 24, and 48 hr after γ radiation, using semiquantitative RT-PCR. The cells overexpressed both p53 and p53DINP1 (left) or p53 alone (right). When analyzed by Western blotting (bottom), overexpression of both genes strongly induced phosphorylation at Ser46 in response to γ radiation. (B) Cell cycle analysis of MCF7 cells overexpressing both p53 and p53DINP1 (left) or p53 alone (right), 72 hr after γ radiation or exposure to adriamycin. The entire cells with and without the expression vectors were subjected to FACS analysis. (C) TUNEL analysis of MCF7 cells overexpressing both p53 and p53DINP1 (left) or p53 alone (right), 72 hr after γ radiation. The orange color represents the nuclear staining by propidium iodide (PI), which shows living cells. The green color represents the nuclear staining by in situ terminal transferase-mediated dUTP nick end labeling, which shows apoptotic cells Molecular Cell 2001 8, 85-94DOI: (10.1016/S1097-2765(01)00284-2)

Figure 6 p53DINP1 Interacts with the Ser46 Kinase for p53 (A) In vitro p53-Ser46 phosphorylation assay. The Ser20, Ser33, and Ser46 kinase activity of whole-cell lysates isolated from irradiated MCF7 cells at 12 hr (lane 1), which had not been immunoprecipitated with anti-p53DINP1 antibody, were shown as positive controls for in vitro p53-Ser20, p53-Ser33, and p53-Ser46 phosphorylation assay. The immune complex was precipitated with anti-p53DINP1 antibody from cell extracts isolated from MCF7 cells not damaged (lane 2, control) or damaged by 30 Gy γ irradiation at 2 hr (lane 3), 4 hr (lane 4), 8 hr (lane 5), and 12 hr (lane 6). The nonspecific protein complex was precipitated with rabbit preimmune IgG (control IgG) from cell extracts isolated at 12 hr (lane 7) from MCF7 cells damaged by 30 Gy γ irradiation. The Ser20 (top), Ser33 (middle), and Ser46 (bottom) phosphorylation activity were shown by Western blotting with anti-Ser20 (α-p-Ser20), anti-Ser33 (α-p-Ser33), and anti-Ser46 (α-p-Ser46) antibodies, respectively. The GST-p53 protein (bottom) was shown by Western blotting with anti-p53 antibody (DO-1). (B) Hypothetical mechanism of p53-dependent apoptosis Molecular Cell 2001 8, 85-94DOI: (10.1016/S1097-2765(01)00284-2)