Transcription activity is required for p53-dependent tumor suppression

Abstract

As a transcription factor, the critical tumor suppressor, p53, directly regulates the transcription of hundreds of genes, leading to cell-cycle arrest, apoptosis, cellular senescence and differentiation. Although it has been assumed that p53 transcription activity is critical for tumor suppression, this assumption has been increasingly contested by recent findings of transcription-independent roles of p53 in apoptosis as well as findings that none of the mutant mice lacking important p53 transcription targets are cancer prone. On the basis of previous findings that p53 transcription activity is abolished in p53(QS) (Leu25Trp26 to Gln25Ser26) knock-in mouse cells after DNA damage, to determine the importance of transcription activity of p53 in tumor suppression, we generated knock-in mice that can conditionally express p53(QS) protein in a Cre-dependent manner. By breeding the knock-in mice with Lck-Cre transgenic mice that specifically express Cre in thymocytes, we show that p53-dependent suppression of thymic lymphomas is abolished in thymocytes expressing high levels of p53(QS) protein. In addition, p53(QS) protein is accumulated in some of the thymic tumors. Therefore, p53 transcription activity induced by DNA damage is required for tumor suppression. Together with the findings that the disruption of various p53-dependent functions individually fails to promote cancer, our findings indicate that various transcription-dependent functions of p53 must collaborate to efficiently suppress tumorigenesis.

Figure 1

Expression and function of p53 in the thymocytes of p53^QSL mice. (a) The p53^QS-Neo knock-in allele. The exons are represented by open boxes. PCR primers to screen for the LoxP/Cre-mediated deletion are indicated by arrowheads. The QS mutations are indicated by an asterisk. The generation of the knock-in ES cells were described previously (Chao et al., 2000). (b) Expression of p53 protein in the thymocytes of Lck-Cre⁺, p53^QSL and Lck-Cre⁺p53^LoxP/LoxP mice before and 5 hours after IR (10Gy). Lck-Cre⁺ mice express the Cre enzyme specifically in the thymocytes but not in other cell types (Lee et al., 2001). In p53^LoxP/LoxP mice, the exons 2–10 of the p53 gene are flanked by LoxP sites and can be deleted from the genome in a Cre-dependent manner (Jonkers et al., 2001). Protein extracts from 2×10⁶ thymocytes were resolved on 10% SDS PAGE gel and transferred to nitrocellulose membrane, which was probed with a polyclonal phosphor-specific antibody against p53 phosphorylated at Ser18 (Cell Signaling Technology, Danvers, MA) or ploclonal antibody against p53 or β-actin (Santa Cruz Biotechnology). The membrane was subsequently probed with a horseradish peroxidase-conjugated secondary antibody and developed with Supersignal Pico reagents (Thermo Scientific, Rockford, IL). (c) Induction of p53 protein levels in Lck-Cre⁺ and p53^QSL thymocytes 8 hours after treatment with increasing concentration of doxorubicin. (d) Expression of p53 protein in the small intestine and thymus of Lck-Cre⁺, p53^QSL and Lck-Cre⁺p53^LoxP/LoxP mice 1 hour after whole body IR (10Gy). No p53 protein can be detected in the brain and liver of these mice of all genotypes by Western blotting (data not shown). (e) Induction of p53 target genes in Lck-Cre⁺ and p53^QSL thymocytes 5 hours after 10Gy of IR. Total RNA from thymocytes was isolated using Trizol (Invitrogen, Carlsbad, CA) and RNAeasy Mini Kit (Qiagen, Valencia, CA), reverse-transcribed using Superscript II RT (Invitrogen). Real-time PCR was performed with an AjBI Prism 7000 (Applied Biosystems, Foster City, CA) with Power SyberGreen PCR Master Mix (Applied Biosystems, Foster City, CA). The PCR conditions were: 10 min at 95°C, 40 cycles of 15 sec at 95°C and 1 min at 60°C. The average Ct value for each gene was determined from triplicate reactions and normalized with the levels of GAPDH as previously described (Chao et al., 2006a). The primers were described previously (Chao et al., 2006a; Lin et al., 2005). Mean value from three independent experiments are presented with standard derivation. (f) p53-dependent apoptosis of Lck-Cre⁺, p53^QSL and Lck-Cre⁺p53^LoxP/LoxP thymocytes 10 hours after increasing dosages of IR. Single cell suspension of thymocytes derived from 4-week-old mice was cultured in DMEM supplemented with 5% FBS and 25 mM HEPES at pH 7 at a density of 10⁶ cells/ml. The cells were irradiated and the percentage of apoptotic cells was analyzed 10 hr later by staining with Annexin V-FITC as described (Chao et al., 2006a).

Figure 2

Tumorigenesis of p53^QSL mice. (a) Survival curve of Lck-Cre⁺, p53^LoxP/LoxPLck-Cre⁺ and p53^QSL mice. N shows the number of mice monitored. The P value for the difference of survival between p53^LoxP/LoxPLck-Cre⁺ and p53^QSL mice is 0.46. (b) Tumor spectrum of p53^LoxP/LoxPLck-Cre⁺ and p53^QSL mice. The p values are shown.

Figure 3

Metastatic phenotypes of thymic tumors in p53^QSL mice. (a) p53 protein is accumulated to high levels in some of the thymic tumors derived from p53^QSL mice. The metastatic and non-metastatic tumors are indicated. (b) The relative expression of p53 target genes in the tumors expressing p53^QS versus in pre-tumor p53^QSL thymocytes. The mRNA levels of each gene were determined by quantitative real time PCR and standardized by the mRNA levels of GAPDH. Mean value from three tumors are presented with standard derivation. (c) Representative histological image of one metastatic thymic tumor in p53^QSL mice that invade the skeleton muscles of the rib cage as well as the one from p53^LoxP/LoxPLck-Cre⁺ mice. Tumor samples were fixed in 10% buffered formalin, embedded in paraffin and sliced. All sections were stained with hematoxylin and eosin for histological assessment as previous described (Chao et al., 2006a).