TRIM8 modulates p53 activity to dictate cell cycle arrest

© 2012 Landes Bioscience.

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www.landesbioscience.com Cell Cycle 1

Cell Cycle 11:3, 1-13; February 1, 2012; © 2012 Landes Bioscience REPORT REPORT

*Correspondence to: Apollonia Tullo and Giuseppe Merla; Email: [email protected] and [email protected]: 10/10/11; Revised: 12/12/11; Accepted: 12/12/11http://dx.doi.org/10.4161/cc.11.3.19008

Introduction

Inactivation of the p53 pathway is a common feature of can-cer cells.1 Intrinsic and extrinsic cellular stresses, DNA dam-age or abnormal proliferative signals act upon the p53 pathway activating different enzymes that modify p53 protein, mainly through phosphorylation, acetylation, ubiquitination, ned-dylation or sumoylation. Some of these post-translational modifications, which reflect different types of stress, lead to p53 stabilization by increasing the protein half-life and activat-ing the ability of p53 to bind the DNA in a sequence-specific manner promoting transcription of genes involved in cell cycle arrest, cellular senescence or apoptosis.2,3 For the full activa-tion of specific groups of target genes, p53 requires the exact combination of post-translational modifications and cofactors recruitment. The deactivation of p53 is also a tightly regulated process. Ubiquitin-proteasome degradation plays a major role in the regulation of p53, predominantly by the E3 ubiquitin

p53 is a central hub in controlling cell proliferation. To maintain genome integrity in response to cellular stress, p53 directly regulates the transcription of genes involved in cell cycle arrest, DNA repair, apoptosis and/or senescence. An array of post-translational modi!cations and protein-protein interactions modulates its stability and activities in order to avoid malignant transformation.

However, to date, it is still not clear how cells decide their own fate in response to di"erent types of stress.Here we describe that the human TRIM8 protein, a member of the TRIM family, is a new modulator of the p53-me-

diated tumor suppression mechanism. We show that under stress conditions, such as UV exposure, p53 induced the expression of TRIM8, which, in turn, stabilized p53, leading to cell cycle arrest and reduction of cell proliferation through enhancement of CDKN1A (p21) and GADD45 expression. TRIM8 silencing reduced the capacity of p53 to activate genes involved in cell cycle arrest and DNA repair in response to cellular stress. Concurrently, TRIM8 overexpression induced the degradation of the MDM2 protein, the principal regulator of p53 stability. Co-immunoprecipitation experiments showed that TRIM8 physically interacted with p53, impairing its interaction with MDM2.

Altogether, our results reveal a previously unknown regulatory pathway controlling p53 activity and suggest TRIM8 as a novel therapeutic target to enhance p53 tumor suppressor activity.

TRIM8 modulates p53 activity to dictate cell cycle arrest

Mariano Francesco Caratozzolo,1,† Lucia Micale,2,† Maria Giuseppina Turturo,2,‡ Silvia Cornacchia,1,3 Carmela Fusco,2 Flaviana Marzano,1,§ Bartolomeo Augello,2 Anna Maria D’Erchia,3 Luisa Guerrini,4 Graziano Pesole,3,5 Elisabetta Sbisà,1 Giuseppe

Merla2,* and Apollonia Tullo1,*

1Institute for Biomedical Technologies; National Research Council; Bari, Italy; 2Medical Genetics Unit; IRCCS Casa Sollievo della So"erenza; San Giovanni Rotondo; Foggia, Italy; 3Department of Biochemistry and Molecular Biology “E. Quagliariello”; University of Bara “A. Moro”; Bari, Italy; 4Department of Biomolecular Sciences and Biotechnologies;

University of Milano; Milano, Italy; 5Institute of Biomembranes and Bioenergetics; National Research Council; Bari, Italy

†These authors contributed equally to this work.

Current addresses: ‡Telethon Institute of Genetics and Medicine; Napoli, Italy; §Department of Biomedicine of Developmental Age; University of Bari “A. Moro”; Bari, Italy

Key words: p53, tumor suppressor, stress response, cell fate, TRIM8

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ligase MDM2 that maintains p53 at low levels in unstressed cellular conditions.4,5

It has been established that MDM2 can differentially cata-lyze either monoubiquitination or polyubiquitination of p53 in a dosage-dependent manner.4 Low dosage of MDM2 induces p53 monoubiquitination, which greatly promotes its mitochondrial translocation and apoptosis,6 whereas high levels promote polyu-biquitination and degradation of p53.

In turn, MDM2 function is supported by several cofactors, like UBE4B and WIP1, which are essential for promoting p53 polyubiquitination and degradation.7,8 Moreover, a number of proteins play an important role in p53 regulation through their interactions with MDM2. They indirectly control p53 activity by changing MDM2 subcellular localization, protein levels or enzymatic function.9-12

Recently, it has been demonstrated that E4F1 behaves as an atypical ubiquitin E3 ligase, which promotes the p53 oligo-ubiq-uitination on lysine residues (K319–K321) distinct from those


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2 Cell Cycle Volume 11 Issue 3

or knockdown did not affect p53 mRNA expression (Figs. 1B and S2A). Conversely, TRIM8 overexpression induced the stabilization of endogenous as well as transfected p53 protein, enhancing its half-life from ~30 to ~120 min, while TRIM8 ablation reduced p53 protein levels (Figs. 1C and D, S2B and C). Similar results were obtained in different cell lines (Fig. S2D). Interestingly, the stabilization of p53 levels was paralleled by an increase of Ser-15 and Ser-20 phosphorylated p53 level, markers of the activation status of p53 for cell cycle arrest activ-ity, but not of Ser-46, linked to p53 apoptotic activity (Figs. 1C and S2B).29-31

MTT proliferation assay and western blot analyses dem-onstrated that the three different TRIM8 constructs (FLAG-TRIM8, HA-TRIM8 and EGFP-TRIM8) equally induced a reduction of cell proliferation and p53 stabilization (Fig. S3).

Consistently, the immunochemical determination of cyto-plasmic histone-associated-DNA fragments demonstrated that p53 overexpression induced apoptosis in HCT116 cells in a dose-dependent manner, but when p53 was co-transfected with TRIM8, the number of apoptotic cells significantly decreased (Fig. 2A). In addition flow cytometric analysis revealed that HCT116 cells overexpressing p53 or TRIM8 yielded a 26% and 19% increase in G1 phase, respectively, compared with control cells (Fig. 2B). The effect resulted amplified in cells overexpress-ing both p53 and TRIM8 (41% increase in G1 phase with respect to control cells) (Fig. 2B, left part). On the contrary, TRIM8 overexpression in HCT116 p53-null cells did not lead to G1 arrest (Fig. 2B, right part). However, in these cells, the ectopic co-expression of p53 and TRIM8 induced a consistent G1 arrest (Fig. 2B, right part). Together, these results demonstrated that the suppression of cell proliferation upon TRIM8 overexpression is due to a p53-dependent cell cycle arrest.

The increased levels of p53 phosphorylated on Ser-15 and Ser-20 and the p53-mediated cell cycle arrest, prompted us to investigate whether TRIM8 might regulate the p53-dependent transcriptional activity of genes involved in cell cycle arrest and related pathways. To assess this hypothesis, we monitored by qRT-PCR, the expression of different p53 transcriptional targets in HCT116 p53wt and in HCT116 p53-null cell lines (Fig. 3A) upon TRIM8 overexpression or silencing. As shown in Figure 3A, TRIM8 overexpression increased the mRNA levels of p21 and GADD45 cell cycle arrest genes (4.3- and 2.2-fold respectively) in HCT116-p53wt but not in HCT116-p53(-/-) cell lines, while the expression levels of the apoptotic genes BAX and PUMA were not affected in both cell lines. Conversely, TRIM8 silencing, achieved using four different specific TRIM8-shRNA, inhibited p21 and GADD45 mRNA expression in the HCT116-p53wt cell line. Similar results were observed in different cell lines (Fig. S4). These data were strongly supported by reporter assay experiments (Fig. 3B), which demonstrated that the ability of p53 to transac-tivate p21 and GADD45 p53 responsive elements (p53REs) was significantly increased when p53 was co-expressed with TRIM8. On the contrary, p53-mediated activation of the BAX promoter was reduced by TRIM8 co-expression. Altogether, these experi-mental evidences demonstrate that TRIM8 activates p53 toward a specific cell cycle arrest program.

targeted by MDM2, resulting in the p53-dependent transcrip-tional activation of target genes involved in growth arrest.13

Several ubiquitin-like proteins (UBL) have been identified, that are not necessarily associated with proteosomal degrada-tion. In particular, p53 function is regulated by at least two UBL proteins: SUMO-1 and NEDD8.5,14,15 p53 sumoylation is mediated by different SUMO-E3-ligases, like Topors,16 PIAS17 and several TRIM proteins,18 and it has been reported to have distinct functional effects, as it is involved in the regulation of subcellular transport, transcriptional activity and cell cycle con-trol. Moreover, it has been shown that MDM2 can also promote the conjugation of SUMO-1 and SUMO-2/3 to p53.19 Even ned-dylation of p53 is mediated by MDM2 and determines the inhi-bition of its transcriptional activity.20

Here, we demonstrate that the human TRIM8 protein is a new modulator of the p53-mediated tumor suppression mechanism.

TRIM8 is a member of the tripartite motif (TRIM, also known as RBCC) protein family defined by the presence of a RING domain, one or two B-boxes and a coiled-coil region.21,22 Alterations of these proteins cause pathological conditions that range from Mendelian disease to cancer development and viral infection.23-27

Recently in a transcriptome-wide analysis of larynx squamous cell carcinoma (LSCC), TRIM8 protein has been associated with metastatic progression. Furthermore, stable expression of TRIM8 in U2OS cells reduced the colony-formation capacity of these cells.28

In this report, we provide experimental evidence that TRIM8 is a p53 direct target gene that induces p53 stabilization and pro-motes the degradation of MDM2, directing the p53 response toward growth arrest and not apoptosis, suggesting that TRIM8 plays an important role in the decision of cell life or cell death controlled by p53.

Results

TRIM8 induces p53 activation, p53-dependent cell cycle arrest and MDM2 degradation. We tested whether the effect of growth suppression mediated by TRIM8, observed by others in LSCC,28 was dependent on p53 tumor suppressor activity. To this end, we investigated the effect of TRIM8 overexpression or knockdown on cellular proliferation rate in different TRIM8-expressing cell lines with p53 wild-type and in p53-null cells (Fig. 1A). The levels of exogenously expressed TRIM8 protein and abrogation of TRIM8 endogenous expression were confirmed by western blotting and qRT-PCR, respectively (Fig. S1). We observed sup-pression of cell proliferation in p53wt cell lines (MCF-7, HCT116 and U2OS) transfected with TRIM8, while no cell cycle per-turbations were detected in absence of p53 (H1299, HCT116 p53-/- and Saos-2) (Fig. 1A). Conversely, silencing of TRIM8 by specific short hairpin RNA (shRNA) resulted in increased cell proliferation rate in p53wt cells but not in p53-null cells (Fig. 1A). Overall, these data support the finding that the effect of TRIM8 on suppression of cell proliferation was p53-dependent.

Next, we analyzed whether TRIM8 is able to modulate p53 mRNA and protein levels. We found that TRIM8 overexpression


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and 4B and C); indeed, while p53 protein half-life increased from ~30 to ~120 min (Figs. 1D and S2C), MDM2 protein half-life decreased from ~120 to 30 min (Fig. 4C). As expected, TRIM8 ablation increased MDM2 protein levels (Fig. 4B).

To further investigate the mechanism of TRIM8 in p53 stabili-zation, we examined whether TRIM8 directly interacted with p53 and/or MDM2. Co-immunoprecipitation experiments showed

As p53 protein levels are mainly regulated by MDM2,4 we assessed whether TRIM8 stabilizes p53 as a results of changes in MDM2 mRNA and/or protein stability. qRT-PCR demonstrated that TRIM8 overexpression or knockdown did not significantly affect MDM2 mRNA expression (Fig. 4A). Notably, we found that, in an exactly opposite manner, TRIM8 overexpression decreased MDM2 protein levels but increased p53 (Figs. 1C and D

Figure 1. Trim8 induces p53 stabilization. (A) Cell proliferation was measured by MTT reduction in the indicated cell lines 72 h after transfection with pcDNA3-FLAG control vector, pcDNA3-FLAG-TRIM8, unspeci!c shRNA (control) or four speci!c TRIM8-shRNAs. (B) qRT-PCR of p53-mRNA in HCT116-p53wt cells transfected with pcDNA3-FLAG control vector, pcDNA3-FLAG-TRIM8, unspeci!c shRNA (control) or 4 speci!c TRIM8-shRNAs. (C) HCT116-p53wt cells were transfected as indicated and immunoblotted as shown. Control HCT116 cells were irradiated with apoptotic UV doses (80 J/m2). Western blot of Actin was conducted as control. (D) p53 half-life was measured by treating HCT116-p53wt cells with cycloheximide (CHX) for the indicated time.


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Figure 2. Trim8 induces p53-dependent cell cycle arrest. (A) In HCT116-p53wt transfected for 72 h with the indicated control or recombinant vectors, apoptosis was measured by immunochemical determination of cytoplasmic histone-associated-DNA fragments. The levels of exogenously expressed proteins were controlled by western blotting analysis. The level of Actin was evaluated as control. (B) Flow cytometric analysis of HCT116-p53wt and HCT116-p53(-/-) cells transfected with the indicated control and recombinant vectors. The levels of exogenously expressed proteins were controlled by western blotting analysis. The level of Actin was evaluated as control.


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whether p53 directly targets TRIM8 expression. We queried the p53FamTag database and identified one putative p53 responsive element (RE) composed of three decamers in the first intron of TRIM8 (Fig. 5A).32 The in vivo binding of p53 to this p53RE was verified by chromatin immunoprecipitation (ChIP) analy-sis and luciferase reporter assays, which demonstrated that this p53RE is functionally active for p53 (Fig. 5B and C). The activa-tion was dependent on functional p53, since the transactivation-defective mutant p53R175H was unable to bind and activate

that TRIM8 physically bound p53 (Fig. 4D) but not MDM2 (Fig. 4E). It is noteworthy that TRIM8 overexpression hardly impaired the interaction between p53 and MDM2 (Fig. 4E).

These data suggest that TRIM8-mediated p53 stabilization may occur through MDM2, although without a direct interac-tion (Fig. 4D and E).

TRIM8 is a direct p53 target gene. It is known that p53 controls the expression of many proteins involved in the regula-tion of its own stability and activity; hence, we sought to assess

Figure 3. TRIM8 induces p53-dependent activation of cell cycle arrest genes. (A) qRT-PCR of the indicated p53 target genes in HCT116-p53wt and in HCT116-p53-/- cells transfected for 72 h with pcDNA3-FLAG control vector, pcDNA3-FLAG-TRIM8, unspeci!c shRNA (control) or 4 speci!c TRIM8-shRNAs. (B) Luciferase assay. p53-null H1299 were co-transfected with pcDNA3 control vector, pcDNA3-p53, pcDNA3-p53 plus pcDNA3-HA-TRIM8 or pcDNA3-HA-TRIM8 with pGL3-basic-luciferase reporter constructs containing p21, GADD45 and BAX p53REs. The levels of exogenously expressed proteins in H1299 cell line were controlled by WB. Cells were lysed and luciferase expression was determined as described in reference 38. Transfection e#cacy was normalized by renilla luciferase activity. Columns, average of at least three independent experiments; bars, SE.


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Figure 4. TRIM8 promotes the degradation of MDM2 and directly interacts with p53 impairing MDM2 binding to p53. (A) qRT-PCR of MDM2-mRNA in HCT116-p53wt cells transfected with pcDNA3-FLAG control vector, pcDNA3-FLAG-TRIM8, unspeci!c shRNA (control) or 4 speci!c TRIM8-shRNAs. (B) HCT116-p53wt cells were transfected as indicated and immunoblotted as shown. Western blot of Actin was conducted as control. (C) MDM2 half-life was measured by treating HCT116-p53wt cells with cycloheximide (CHX) for the indicated time. Western blot of Actin was conducted as control. (D) Co-immunoprecipitation of p53 and TRIM8. HCT116p53ko cell lysates were subjected to immunoprecipitation with p53 speci!c antibody DO-1. The immunoprecipitated complexes were analyzed by immunoblotting using FLAG antibody. (E) Co-immunoprecipitation of MDM2 and TRIM8. HCT116p-53ko were co-transfected with the indicated plasmids. Cell lysates were immunoprecipitated with anti-MDM2 or anti-p53. The immunoprecipitated complexes were analyzed by immunoblotting with the indicated antibodies.


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TRIM8 transcription, while p53 silencing decreased TRIM8 expression level in MCF-7 cells (Fig. 5D and E).

TRIM8-RING domain is essential to regulate p53 and MDM2 stability and activity. To identify the TRIM8 region responsible for p53 activation, we transfected MCF-7 cells with

the reporter constructs (Fig. 5B and C). Moreover, p53REmut-TRIM8, carrying a deletion of one decamer out of three (Fig. 5C), was weakly responsive to wt p53. Consistently, in 293 T-rex cells stably transfected with p53 under the control of a tetracy-cline-inducible promoter, p53 overexpression positively regulated

Figure 5. TRIM8 is a direct p53 target gene. (A) Schematic map of the human TRIM8 genomic region containing the putative p53RE (TRIM8-p53RE) with the related sequence. (B) In vivo recruitment of p53 and p53R175H mutant to TRIM8-p53RE present in the TRIM8 gene by Chromatin-immuno-precipitation assay. DNA fragments were analyzed by PCR using speci!c primers. Primers speci!c for the unrelated interleukin-10 promoter were used as negative control. (C) Luciferase assay. E"ect of p53 and p53R175H mutant on the transcriptional activity of TRIM8-p53RE and of TRIM8-p53REmut. p53-null H1299 cells were co-transfected with plasmids expressing p53 or its mutated forms (p53R175H) and pGL3-Basic-TRIM8-p53RE or pGL3-Basic-TRIM8-p53REmut luciferase reporter. The luciferase activities were measured 48 h later. The data represent the mean of three independent experi-ments. The levels of exogenously expressed protein were controlled by western blotting. (D) TRIM8 qRT-PCR in 293-Trex cell line stably transfected with control vector or the p53 cDNA or the p53H175 cDNA (negative control). The levels of exogenously expressed protein were controlled by western blotting. (E) TRIM8 qRT-PCR in MCF-7 cell line transfected with unspeci!c scramble RNA or speci!c p53-siRNA. The level of endogenous p53 protein was controlled by western blotting.


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to UV radiation, a stress condition in which p53 is induced. To this end, in p53wt and p53-null HCT116 cells, TRIM8 was silenced by four specific short hairpins and, subsequently, cells were exposed to 20 J/m2 and 80 J/m2 UV light in order to induce cell cycle arrest and apoptosis, respectively, as demon-strated by the cytofluorimetric analyses (Fig. 7A). By perform-ing qRT-PCR, we found that the endogenous TRIM8 mRNA levels were increased only in p53wt HCT116 cells when they were arrested in G1 (after 20 J/m2 UV light irradiation) and much less when the cells were in apoptosis (after 80 J/m2 UV light irra-diation) (Fig. 7C, TRIM8). This experiment demonstrated that TRIM8 expression was p53-dependent after DNA damage that induced cell cycle arrest.

different TRIM8 deletion mutants (Fig. 6A). Interestingly, as shown by MTT cell proliferation assays, western blots and luciferase reporter assays, only TRIM8 mutant protein lacking the RING domain (TRIM8-!RING) was unable to suppress cell proliferation, promote p53 stabilization and activation and MDM2 degradation (Fig. 6B). Consequently, TRIM8-!RING mutant was not able to induce the expression of p53 direct tar-get genes involved in cell cycle arrest (i.e., p21) and DNA repair (i.e., GADD45) (Fig. 6C).

Overall these data demonstrated that the RING domain is necessary to modulate MDM2 and p53 stability and activity.

TRIM8 silencing prevents p53 activation after UV radia-tion. Finally, we tested the effects of TRIM8 ablation in response

Figure 6. TRIM8-RING domain is essential to modulate p53 and MDM2 stability. (A) TRIM8 deletion mutants used in proliferation and reporter stud-ies are shown with the related molecular weight (kDa). R, RING-!nger motif; B1, B2, B boxes; CC, coiled-coil domain; RFP, C-terminal domain. (B) Cell proliferation was measured by MTT reduction in the MCF7 cell line transfected for 72 h with pcDNA3-HA control vector, pcDNA3-HA-TRIM8 or with TRIM8-deletion mutants, lacking the two B-boxes (TRIM8-!BB), the coiled coil domain (TRIM8-!CC), the RING domain (TRIM8-!RING), or the RFP domain (TRIM8-!RFP). At the same time, the levels of p53 and MDM2 proteins were assessed by immunoblotting with the indicated antibodies. (C) Luciferase assay. p53-null H1299 were co-transfected with pcDNA3 control vector, pcDNA3-p53, pcDNA3-p53 and pcDNA3-HA-TRIM8, or pcDNA3-HA-TRIM8 deletion mutants, with pGL3-basic-luciferase reporter constructs containing p21, GADD45 or BAX p53REs. The levels of exogenously expressed proteins in H1299 cells were controlled by WB. Cells were lysed and luciferase expression was determined as described in reference 38.


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Figure 7. TRIM8 silencing prevents p53 stabilization and activation after UV exposure. (A) HCT116-p53wt and HCT116-p53-/- cells were transfected with pRS control vector or 4 speci!c TRIM8-shRNAs and exposed to UV treatment (24 h) as indicated. Flow cytometric analysis was performed to monitor-ize the cycle phase of the cells. (B) The proteins were immunoblotted as indicated. (C) qRT-PCR was performed to detect TRIM8, p21, BAX and GADD45 transcript levels.


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to p53 apoptotic activity (Figs. 1C and 7B). Consequently, these molecular events lead to p53-dependent transcriptional activa-tion of p21 and GADD45 but not of PUMA and BAX promoters (Fig. 3A and B).

Most importantly, we found that after a genotoxic stress, such as UV irradiation, in cells with TRIM8 deficit, MDM2 protein levels are stabilized and, accordingly, p53 stabilization is impaired (Fig. 7B). As a consequence, in these cells, a reduced p53-dependent transcriptional activation of growth arrest direct target genes (p21 and GADD45) was detected (Fig. 7C).

We found that the RING domain of TRIM8 is responsible for both p53 stabilization/activation and MDM2 degradation (Fig. 6). TRIM8 is a member of the TRIM family proteins, and it has been reported that many of them act as E3-ubiquitin ligases through their RING domain to target specific substrates for proteasome-mediated degradation.21,25,33 Indeed, ubiquitina-tion regulates a diverse spectrum of cellular processes by provid-ing a specific signal for intracellular protein degradation as well as some degradation-independent functions. Recently, it has been shown that p53 could also be modified by ubiquitination with-out undergoing degradation. Indeed, MDM2 itself can differ-entially catalyze either p53 polyubiquitination targeting p53 to degradation or p53 monoubiquitination promoting p53 nuclear export.4 Moreover, the atypical ubiquitin ligase E4F1 catalyzes p53 ubiquitination at Lys320, which recruits p53 to chromatin. Interestingly, this coincides with p53-mediated growth arrest but not apoptosis,13 demonstrating how ubiquitination is capable of modifying p53’s biological response in decision-making: growth arrest vs. apoptosis.

Notably other TRIMs are involved in p53 regulation.34 For instance, the RING domain of TRIM24 functions as an E3-ubiquitin ligase that targets p53 for degradation, and its depletion induces p53-dependent apoptosis.34,35 Yet, the pro-myelocytic leukemia protein PML/TRIM19 is a p53 target that facilitates p53-Thr18 phosphorylation in response to DNA dam-age by recruiting p53 into PML nuclear bodies, thereby leading to p53 activation by protecting it from MDM2 inhibition.36 More recently, the ataxia telangiectasia group D-complementing, ATDC/TRIM29 protein has been shown to bind and antagonize p53-mediated functions.37

Collectively our experiments demonstrate the existence of a new feedback loop in the tumor suppressor circuit controlled by p53 with TRIM8 as a new key functional modulator of p53 sta-bility and activity that directs the cells toward a program of cell cycle arrest.

A more detailed dissection of TRIM8-mediated regulation of p53 activity or their interacting partners will significantly improve our understanding of the sophisticated framework based on p53 dynamics and will provide deeper insight into develop-ment of therapeutic approaches to suppress the oncogenic growth in human tumors by restoring p53 activity.

Materials and Methods

Cells and treatments. All the cell lines were cultured in Dulbecco’s modified Eagle’s medium (DMEM) plus 10% fetal bovine serum

Interestingly, in the same cells, western blotting experiments demonstrated that TRIM8 silencing impaired p53 stabiliza-tion, p53 phosphorylation on Ser-15 and Ser-20 (Fig. 7B) and p53-dependent transcriptional activation of p21 and GADD45 (Fig. 7C). Additionally, although TRIM8 silencing decreased p53 stabilization after apoptotic UV doses (80 J/m2 UV) (Fig. 7B), it did not affect p53 phosphorylation on Ser-46, and, conse-quently, the transcriptional activation of the p53-pro-apoptotic target gene BAX was not compromised (Fig. 7C). Similar results were obtained in p53wt MCF-7 cells and in p53-null H1299 cells (Fig. S5A and B). Consistent with p53 stabilization after UV irradiation, the HCT116 p53wt cells showed a decrease in MDM2 protein levels, which instead recovered in the same cells with TRIM8 deficit where p53 stabilization is impaired (Fig. 7B, left part). This data was also confirmed in the p53wt MCF-7 cell line (Fig. S5A).

Altogether, these results demonstrate that TRIM8 silencing impairs only p53 activation toward a cell cycle arrest program after a genotoxic stress without interfering in the p53-mediated apoptotic pathway.

Discussion

The p53 tumor suppressor gene family members have a central role in the intricate signaling network controlling cell life and death. The p53 protein is a promising target in fighting cancer because of its high power to mediate tumor suppression, includ-ing irreversible growth suppression and induction of apoptosis, inhibition of angiogenesis as well as the blockade of cellular inva-sion and metastasis. Approximately 50% of all human tumors carry point mutations in the p53 gene that transform the p53 tumor suppressor gene in a dominant oncogene. In tumors with-out p53 mutations (the remaining 50%), p53 is often rendered functionally inert by the inactivation of its positive modula-tors or by the activation of its negative factors, which block the p53 transcriptional activity. The stabilization and activation of wild-type p53 are thus crucial to prevent cells from becoming cancerous.

Therefore, one of the most important challenges is the discov-ery of additional p53-inactivating pathways that might account for escape from p53 control in malignancy with wild-type p53.

With this study, we demonstrated that TRIM8 is a novel p53 direct target gene (Fig. 5A–E) and is an important component in controlling the molecular switch that directs p53 toward tran-scriptional activation of cell cycle arrest genes, such as p21 and GADD45 (Fig. 3A and B). At the same time, TRIM8 induced the degradation of MDM2, the main negative regulator of p53 stability (Fig. 4).

Moreover, we show that under stress conditions, such as UV treatment, p53 promotes transcription of TRIM8 (Fig. 7C), which, in turn, interacting with p53, induces its stabilization and p53-dependent cell growth arrest. Notably, the interaction between TRIM8 and p53 impaired the binding of MDM2 to p53 (Fig. 4E). The stabilized p53 is phosphorylated on Ser-15 and Ser-20, markers of the activation status of p53 for cell cycle arrest, but not on Ser-46, a post-translational modification linked


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RNA extraction, reverse transcription and real time PCR analysis. Total cellular RNA was extracted using the RNasy mini kit (Qiagen). Reverse transcription reactions were performed using the reverse transcription Archive Kit (Applied BiosystemsTM), according to the manufacturer’s instruction. The real-time PCR reactions were performed on an Applied BiosystemsTM 7900HT, as described by the manufacturer. The reaction mixtures con-tain 2x TM Master Mix Buffer, PDAR System Target 20x FAM (TaqMan® Gene Expression Assays), cDNA template and water. Cycle threshold (Ct) values were obtained graphically, auto-matically by the instrument, for TRIM8, p21 (also known as CDKN1A), BAX, PUMA (also known as BBC3) and GADD45. The glyceraldheyde 3P-dehydrogenase (GAPDH) gene was our internal standard. Reactions without cDNA were included as negative control. The relative gene expression for each experi-ment was calculated using the non-treated sample as a calibrator. The data reported represent the average of at least three indepen-dent experiments and are shown with their standard deviations.

Luciferase assay. The fragments containing the p53-RE of the TRIM8 gene (GeneID: 81603) and the fragments containing the p53-REs of p21 (GeneID: 1026), GADD45 (GeneID: 1647) and BAX (GeneID: 581) genes were amplified from human genomic DNA and cloned upstream of the reporter luciferase gene in the pGL-3 Basic plasmid (Promega).

DNA fragments were amplified by PCR using specific prim-ers. Primer sequences are available upon request.

2 x 105 human p53-null H1299 cells were plated in 6-well plates 24 h before transfection (60–80% confluency).

pGL-3 basic recombinant vectors with the expression vec-tor containing wt p53, or its mutant version p53R175H, plus pRLSV40 (Promega) were transfected for the study of the p53REs present in the regulatory regions of TRIM8 gene.

pGL-3 basic recombinant vectors with an expression vector containing wt p53, and/or with an expression vector containing HA-TRIM8 or its mutant deletion versions HA-TRIM8!BB, HA-TRIM8!CC, HA-TRIM8!RING and HA-TRIM8!RFP, plus pRLSV40 (Promega) were transfected for the study of the in vitro action of TRIM8 on the p53REs identified in the p21, BAX and GADD45 p53-target genes.

Transient reporter assays were performed as previously described in reference 38.

Transfection efficiency was determined by renilla activity. The data reported represent the average of at least three independent experiments and are shown with their standard deviations.

Small interfering RNA (siRNAs) transfections. 1 x 105 human MCF-7 cells were plated 24 h before transfection at 60–80% confluency. One hundred microliters of D-MEM with-out serum were incubated with 15 "l CodeBreakerTM siRNA Transfection Reagent (Promega) and incubated at room tempera-ture for 20 min.

Then 80 nM of scramble (control) or p53 small interfering RNAs (DharmaconTM) were added to the medium containing the transfection reagent and incubated at room temperature for 20 min and subsequently added to the cell cultures for 48 h.

Short hairpin RNA (shRNAs) transfections. 5 x 105 human MCF-7 cells were plated 24 h before transfection at 60–80%

(FBS), L-Glutamine (2 mM), penicillin (100 U/ ml) and strepto-mycin (100 "g/ml) at 37°C, 5% CO2.

UV radiation was used at low doses (20 J/m2) and at high doses (80 J/m2) to induce cell cycle arrest or apoptosis, respec-tively. Cicloheximide (CHX) (Sigma) was used at the final con-centration of 100 "g/ml.

Cell proliferation assays by MTT reduction. 1 x 105 cells were plated in six-well plates. After treatments, 200 "l of MTT solution (5 mg/ml) was added to the cells for 4 h at 37°C. Then the supernatant was removed, and the blue formazan crystals were resuspended in isopropanol prior to reading the absorbance at 580 nm.

Protein extraction and western blot analysis. Cells were plated in 100 mm culture dishes at a density of 5 x 105 cells/ml. After treatments, cells were lysed, and proteins were extracted as previously described in reference 38.

For immunoblotting, the following primary antibodies were used: p53 specific DO-1 (Santa Cruz, 1:300), p53-Ser15P (Santa Cruz, 1:100), p53-Ser20P (Santa Cruz, 1:100), p53-Ser46P (Santa Cruz, 1:100), Anti-FLAG (Sigma, 1:1,000), Anti-HA (Bethyl, 1:1,000), Anti-GFP (Santa Cruz, 1:1,000), Anti-Actin Ab-1 antibodies kit (Calbiochem, 1:2,000). Bound primary antibodies were visualized using ECL western blotting or ECL plus western blotting detection reagents (Amersham Pharmacia Biotech).

Co-immunoprecipitation and western blot analysis. Co-immunoprecipitation experiments were performed by lys-ing cells in RIPA buffer addicted with glycerol 10% to stabi-lize protein-protein interactions. Protein complexes were then immunoprecipitated using appropriate antibodies. Complexes were analyzed by western blotting using appropriate antibodies, p53-specific DO-1 (Santa Cruz Biotechnology), anti-MDM2 (Calbiochem, Ab-2 2A10) and anti-FLAG (Sigma, 1:1,000). Horseradish peroxidase conjugated anti-mouse and anti-rabbit antibodies (GE Healthcare) and the ECL chemiluminescence system (GE Healthcare) were used for detection.

Fusion plasmids. The full-length ORF of human TRIM8 was cloned into pcDNA3 vector modified to include either a myc-EGFP, FLAG-tag or HA-tag. TRIM8 deletion mutants were created using appropriate oligonucleotides and amplification fol-lowed by in-frame insertion into the pcDNA3 HA-tag vector.

MTT proliferation assay and western blot analyses dem-onstrated that the three different TRIM8 constructs equally induced a reduction of cell proliferation and p53 stabilization (Fig. S3).

Cell cycle analysis. The total cell population, including float-ing and adherent cells, was harvested, washed twice with 1x PBS, treated with 150 mg/ml RNase A, 5 mg/ml propidium iodide (PI), NP40 0.1% at room temperature for 1 h. The cells were analyzed in a FACScalibur; cell cycle and apoptosis analyses were performed using ModFit analysis software (Becton Dickinson).

Immunochemical determination of cytoplasmic histone-associated DNA fragments. The levels of mono- and oligo-nucleo-somes in the cytoplasmic fraction of cell lysates were determined after the treatments by using the Cell Death Detection ELISAPLUS (RocheTM) according to the manufacturer’s instructions.


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Primer sequences are available upon request. Interleukin-10 pro-moter (IL-10) was used as a negative control.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgements

We thank E. Perlino and V. Calabrò for helpful discussion and critical reading the manuscript.

Financial Support

This work was supported by Progetto Strategico Regione Puglia del. 6.8.2005, n.1171 and by Telethon GGP11097A to Luisa Guerrini and by Italian Ministry of Health (Ricerca Corrente 2008-10) to Giuseppe Merla and by Fonazione Cassa di Risparmio di Puglia (prot. 277/11) to Apollonia Tullo.

Note

Supplemental materials can be found at:www.landesbioscience.com/journals/cc/article/19008

confluency. Two hundred microliters of D-MEM without serum were incubated with Mirus transfection reagent (Tema Ricerca) for 20 min at room temperature.

Five micrograms of empty pRS (control) or of 4 different TRIM8 short hairpin RNAs (OrigeneTM) were added to the medium containing the transfection reagent and incubated at room temperature for 20 min and subsequently added to the cell cultures for 48 h.

Chromatin immunoprecipitation (ChIP) assay. 293 T-rex cells were treated as previously described in reference 39. At the given time points, proteins were crosslinked to DNA in living nuclei and chromatin immunoprecipitation assay was performed as described in reference 38.

Five micrograms of the following antibodies were used: p53 antibody DO-1 (Santa Cruz), acetylated H4-histone anti-body (UpstateTM) or unrelated control anti-Flag antibody (Sigma).

DNA fragments were analyzed by PCR using specific primers for the four p53REs identified in the TRIM8 regulatory regions.

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TRIM8 modulates p53 activity to dictate cell cycle arrest