Activities and response to DNA damage of latent and active sequence-specific DNA binding forms of mouse p53 - PubMed (original) (raw)

Activities and response to DNA damage of latent and active sequence-specific DNA binding forms of mouse p53

Y Wu et al. Proc Natl Acad Sci U S A. 1997.

Abstract

The mouse p53 protein generated by alternative splicing (p53as) has amino acid substitutions at its C terminus that result in constitutively active sequence-specific DNA binding (active form), whereas p53 protein itself binds inefficiently (latent form) unless activated by C-terminal modification. Exogenous p53as expression activated transcription of reporter plasmids containing p53 binding sequences and inhibited growth of mouse and human cells lacking functional endogenous p53. Inducible p53as in stably transfected p53 null fibroblasts increased p21(WAF1/Cip-1/Sdi) and decreased bcl-2 protein steady-state levels. Endogenous p53as and p53 proteins differed in response to cellular DNA damage. p53 protein was induced transiently in normal keratinocytes and fibroblasts whereas p53as protein accumulation was sustained in parallel with induction of p21(WAF1/Cip-1/Sdi) protein and mRNA, in support of p53as transcriptional activity. Endogenous p53 and p53as proteins in epidermal tumor cells responded to DNA damage with different kinetics of nuclear accumulation and efficiencies of binding to a p53 consensus DNA sequence. A model is proposed in which C-terminally distinct p53 protein forms specialize in functions, with latent p53 forms primarily for rapid non-sequence-specific binding to sites of DNA damage and active p53 forms for sustained regulation of transcription and growth.

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Figures

Figure 1

Figure 1

Growth inhibition assays. (A) Saos2 cells were transfected with plasmids containing a neo resistance gene and either no insert, p53, or p53as cDNA. After selection in G418 for 3 weeks, fixed cells were stained with Giemsa. Mean total colony area (mm2) per four 60-mm dishes is shown. The data were consistent in two experiments. (B) Stable (10)1 fibroblast clones containing neo resistance control plasmid, alone or cotransfected with plasmids containing inducible p53as (representative of two clones with integrated p53as), or p53 (four clones), were exposed to CdCl2 (0 hr), and relative cell number was determined as described in Materials and Methods.

Figure 2

Figure 2

Effects on p53 downstream genes in (10)1 cells. (A) Stable inducible p53, p53as, or neo control cell clones were exposed to CdCl2 as in Fig. 1_B_. After 18 hr cells were lysed and 50 μg of protein/lane was immunoblotted using anti-p21 or anti-bcl-2 antibodies. (B) Transactivation of reporter plasmids transiently cotransfected with pCMVp53as, pCMVp53, or pCMV. Cells were lysed after 40–48 hr, and chloramphenicol acetyl transferase (PG13, MG15) or luciferase activity (WWP, WT-30) was measured, standardized to total protein, and expressed relative to pCMV control as 1. Fold activation in three replicate experiments was 3–20 (WWP), 5–18 (WT-30), and 9–15 (PG13).

Figure 3

Figure 3

Response to x-ray (400 rads) of normal (A) 291 epidermal cells or secondary mouse fibroblasts (B) detectable by Western immunoblotting. Proteins were extracted at the indicated times postirradiation and reacted with PAb122 (p53), ApAs (p53as), mp21 (p21), or anti-Hsc70 as loading control. (C) Northern blots. Cells were lysed and 7 μg total RNA per lane was separated in 1.2% formaldehyde agarose, transferred to a membrane, and probed using a radiolabeled fragment of mouse p21/waf1 cDNA. Controls included ethidium bromide staining of rRNA (for loading) and cells exposed to solvent for 4 (4C) or 24 hr (24C).

Figure 4

Figure 4

Response to 60 μM genistein detectable by immunoblotting. 291 cells and derivative tumor cells 291.05RAT (05RAT) were treated for the times indicated, and proteins were extracted and analyzed as in Fig. 3.

Figure 5

Figure 5

p53as and p53 detection by indirect immunofluorescence. 291.05RAT cells were treated with 60 μM genistein for the times indicated, fixed, and immunostained with ApAs (p53as), PAb421 (p53), 6.2 (both), or IgG/preimmune controls (negative, i.e., <1 staining intensity on scale of 1 to 4; data not shown). Positive cells (intensity, 2.5–4) per total cells per coverslip (%) for nuclear/cytoplasmic staining are shown (at 0 hr, 0.1–5% reacted with antibodies). The peak nuclear antibody reactivity for PAb421 (3 hr) and for ApAs (24–48 hr) is representative of six experiments.

Figure 6

Figure 6

Electrophoretic mobility-shift assay. 291.05RAT cells were harvested at the times indicated after treatment with 60 μM genistein. Equal amounts of protein were reacted with a 32P-labeled p53 consensus DNA sequence in the presence or absence of 200 ng of the antibodies indicated and visualized by autoradiography. The positions of migration of baculovirus-purified recombinant human p53 + PAb421 (solid arrow) and mouse p53as (open arrow) are shown. Free probe migrated at twice the distance of the rapidly migrating (unlabeled) nonspecific band and was present in vast excess of the signals shown.

Figure 7

Figure 7

Model: p53 forms respond to DNA damage with distinct kinetics. The p53 forms latent for sequence-specific DNA binding are induced rapidly and specialize in nonspecific DNA binding at sites of DNA damage. Transcription is inhibited through catalysis of strand annealing or by binding to TATA box binding protein (TBP). Forms of p53 active for sequence-specific DNA binding are sustained to evoke long-lasting effects on p53 downstream gene transcription, growth arrest, apoptosis, or cell differentiation. Latent p53 may be activated through posttranslational modifications or binding to single-stranded DNA by-products of nucleotide excision repair, targeting sequence-specific transcriptional effects to sites of intact DNA.

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