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Carcinogenesis Advance Access originally published online on May 29, 2008
Carcinogenesis 2008 29(7):1343-1350; doi:10.1093/carcin/bgm302
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Loss-of-function genetic screening identifies a cluster of ribosomal proteins regulating p53 function

Maria E. Castro, Juan F.M. Leal, Matilde E. Lleonart1, Santiago Ramon y Cajal1 and Amancio Carnero*

Experimental Therapeutics Programme, Centro Nacional de Investigaciones Oncológicas, C/ Melchor Fernández Almagro, 3, 28029 Madrid, Spain
1 Departmento Anatomía Patológica, Hospital Vall d'Hebrón, Barcelona, Spain

* To whom correspondence should be addressed. Tel: +34 91 732 8021; Fax: +34 91 224 6976; Email: acarnero{at}cnio.es


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Supplementary materials
 Funding
 References
 
Introduction of conditional murine p53 (p53val135) and oncogenic ras into double p53/p21-null mouse embryonic fibroblasts (MEFs) showed that p21waf1 was not required for combined ras/p53-induced senescent-like growth arrest. We used this cellular system to identify key players in the ras-p53-induced senescence in the absence of p21. Applying a retroviral-based genetic screen, we obtained mRNA antisense fragments against a cluster of 14 different ribosomal proteins which loss of function bypasses p53-induced growth arrest. The expression of the ribosomal protein antisense fragments reduced the transcriptional activity of p53. Experiments with eGFP-p53 chimeras suggest that the effect is mediated by a reduction of p53. To study whether p53 was downregulated by MDM2-dependent degradation, we tested the effect of the RP antisenses in double p53/MDM2-null MEFs and observed that in the absence of MDM2, reduction of the RP levels also decreases p53 levels. Therefore, although we cannot discard other unknown mechanism, we suggest that the decrease in the levels of ribosomal proteins might inhibit p53-specific translation. Finally, quantitative analysis comparing levels of mRNA in tumours versus mRNA in normal tissue of the same organ and patient showed that a variable percentage of lung, prostate or colon tumours have reduced levels of the RPs tested. Interestingly, in most cases, the reduction of ribosomal protein mRNAs occurs only to 50%. Our data suggest that ribosomal protein imbalance might contribute to p53 regulation through the ribosomal biogenesis checkpoint.

Abbreviations: MEF, mouse embryonic fibroblast; PCR, polymerase chain reaction


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Supplementary materials
 Funding
 References
 
The tumour suppressor p53 is the most commonly mutated gene in human cancer: ~50% of the human tumours carry mutations in this gene (1). Furthermore, considering also those mutations that make inoperative the p53 network, most of the tumours fall in this category (2).

p53 protein exerts its antitumoural function through three main biological activities: cell-cycle arrest (3), induction of apoptosis (4) and induction of senescence (5). p53 is a transcription factor that integrates many cellular stress signals including DNA damage, hypoxia or oncogenic activation (6). Those signals induce the stabilization of p53, normally a very short lived and low abundant protein (7). They also render p53 active, adopting a tetrameric quaternary structure (7,8). Among the p53-transactivated genes, we can underline p21 (that controls the G1/S checkpoint of the cell cycle), 14-3-3{sigma} (that controls the G2/M checkpoint of the cell cycle), Gadd45 (important for DNA repair), bax (that activates the cellular apoptotic programme) or MDM2 (that participates with p53 in a negative feedback loop, controlling the activity, stability and subcellular localization of p53) (9). The outcome of p53 activity can be either reversible cell-cycle arrest or permanent withdrawal from cell proliferation followed by the induction of apoptosis or cellular senescence (5,10,11).

The tumour suppressor activity of p53 is controlled in multiple ways and this control is necessary for the normal outcome of many physiological processes such as cell survival under normal conditions, embryonic development, etc. The main actor is the oncoprotein MDM2, which establishes a negative feedback loop with p53 only broken when the activity of p53 is necessary or when MDM2 is deregulated. MDM2 targets p53 for proteasomal degradation (12,13) through its E3 ubiquitin ligase activity. In up to 10% of human tumours, MDM2 is overexpressed. In all those tumours, the disruption of the negative feedback loop between p53 and MDM2 would be sufficient to generate stable and active p53, thus committing tumour cells to cell-cycle arrest, senescence or apoptosis. Other regulatory mechanisms can be included in this pathway, such as MDM2–ARF binding and sequestration in the nucleolus or MDM2/p53 modification upon cellular stress to avoid protein–protein interaction (14–16), rendering p53 free and active as a transcription factor. Recently, MDMX activation has been also reported downregulating p53 (17).

To search for new p53 regulators or downstream targets that can induce permanent p53-dependent cell-cycle arrest in the absence of p21, we took advantage of the mouse temperature-sensitive p53 (p53val135) that allows conditional and reversible activation of p53. p53 expression in double p53;p21 knockout mouse embryonic fibroblasts (MEFs) is sufficient to induce cell-cycle arrest. When oncogenic ras is also expressed, p53–/–;p21–/–;ts-ras cells arrest at the permissive temperature with all signs of cellular senescence (18). In this system, we have used a systematic approach to select antisense RNA fragments that inhibit the p53 tumour suppressor function (19). We found a cluster of antisense fragments whose enforced expression inhibits p53-induced arrest in MEFs. These antisense codify for 14 different ribosomal proteins.

Ribosome biogenesis consumes a major part of the cell’s energy and resources and plays a key role in the cell’s life cycle (20–22). The entire ribosome, composed of four rRNA species and ~75 ribosomal proteins distributed between two subunits of 40S and 60S, is assembled in the nucleolus. The mature 40S ribosomal subunit contains the 18S rRNA and ~32 ribosomal proteins, whereas the 60S subunit is composed of the 5S, 5.8S and 25/28S rRNAs and ~45 ribosomal proteins (23,24). Proper assembly of each ribosomal subunit requires the coordination of several events, including the synthesis and import of ribosomal proteins to the nucleus, the synthesis and processing of rRNAs and the concomitant assembly of ribosomal proteins into the preribosomal subunits. Conceivably, perturbation of ribosome biogenesis will have a profound effect on the cell, and a tight control system to coordinate cell division with cell growth must exist in all the multicellular organisms. Nonetheless, little is known about how cells coordinate ribosome biogenesis and the cell cycle.

In the present paper, we report the identification of the 14 different ribosomal proteins which loss of function bypasses p53-induced growth arrest and the characterization of its relationship to p53 activity.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Supplementary materials
 Funding
 References
 
Cell culture
Primary MEFs from p53–/– mice and p53–/–p21–/– double knockout mice were derived from day 13.5 embryos as described previously. Cells expressing murine p53val135 were generated by retrovirus-mediated gene transfer of p53val135 into p53–/– MEFs (p53–/–;ts) and p53–/–;p21–/– MEFs (p53–/–;p21–/–;ts cells). Cells expressing val12-ras were generated by retrovirus-mediated gene transfer of pBabepuro-ras(val12) into p53–/– (p53–/– ras), p53–/–;ts (p53–/–;ts-ras) and p53–/–;p21–/–;ts (p53–/–;p21–/–;ts-ras cells). Cells were cultured in Dulbecco’s modified Eagle’s medium (GIBCO) supplemented with 10% foetal bovine serum (Sigma) and 1% penicillin G–streptomycin sulphate (Sigma).

The following retroviral vectors were used: pMARX-IV vector (25) and p53val135-mutant cDNA in pWZLHygro. Retrovirus-mediated gene transfer was performed as described previously (25).

Growth curves
Cells were infected as before. At population doubling 12, 3 x 103 cells were plated in 2.5 cm dishes. At 2- to 3-day intervals, cells were fixed and stained with crystal violet. After extensive washing, crystal violet was re-solubilized in 10% acetic acid and quantitated spectrophotometrically at 595 nm as a relative measure of cell number.

Generation of the antisense library
mRNA was extracted from MEFs terminally arrested at replicative senescence. Aliquots of 2 µg of total mRNA were used for the generation of the library. Randomly primed cDNA fragments of the polyA+ mRNA were synthesized, size selected (50–500 nt) on a S400 column (Pharmacia) and cloned into the EcoRI and XhoI sites of pMARX-IVpuro in the antisense orientation.

Retroviral-mediated antisense library transfer
Samples of 5 x 106 LinXE ecotropic packing cells were plated per 10 cm dish, incubated for 24 h and transfected by calcium phosphate precipitation using 20 µg of retroviral plasmid. After 48 h, the virus-containing medium was filtered (0.45 µm filter; Millipore) and supplemented with 8 µg/ml polybrene (Sigma) and an equal volume of fresh medium. One day before infection, target fibroblasts were plated at 8 x 105 cells per 10 cm dish and incubated overnight. For infections, culture medium was replaced by the appropriate viral supernatant and the culture plates were centrifuged (1 h, 1500 r.p.m.) and incubated at 37°C for 16 h.

In vitro recovery of the proviruses
Total genomic DNA was extracted from cells grown to confluence in a 10 cm plate. It was then treated with RNase A (50 µg/ml, 30 min) and proteinase K (100 µg/ml final concentration) and extracted twice with phenol–chloroform. Following ethanol precipitation, genomic DNA was washed extensively with 70% ethanol and dissolved in 200 µl of ultrapure water.

To excise the proviruses, 10 µg of genomic DNA were digested with CRE recombinase (DNA final concentration 0.1 µg/µl) for 3 h at 37°C, extracted with phenol–chloroform and ethanol precipitated. DNA was washed extensively with 70% ethanol and dissolved in 5 µl of water. Aliquots of 2–5 µg of total DNA were electroporated into DH10B-lac-trfA bacteria and proviruses recovered from zeocin-resistant bacterial colonies.

Temperature shifts and cell proliferation analysis
Cells were seeded at low density (6 x 104 cells/10 cm dish) in two sister plates and grown at either 32 or 39°C. After 15 days in culture, we compared the number of colonies between the plate kept at 32°C and the sister plate grown at 39°C.

Immunoblotting
Cells were washed twice with phosphate-buffered saline, harvested with lysis solution [50 mM Tris pH 7.5; 150 mM NaCl, 1% vol/vol Nonidet P40, CompleteTM protease inhibitor cocktail (Roche)] and centrifuged 10 min at 13 200 r.p.m. to eliminate DNA and lipid debris. The protein concentration in the extract was determined by the method of Bradford (Bio-Rad). The appropriate amount of protein was then denaturized with the same volume of Laemmli 2x buffer and boiled for 3 min. Samples were loaded onto acrylamide/bis sodium dodecyl sulphate gels and subjected to electrophoresis. Proteins were then transferred onto a polyvinylidene difluoride membrane (Immobilon P, Millipore), pre-hybridized with 1% Bovine serum albumin phosphate-buffered saline and hybridized with the primary antibody [p53 (FL-393), sc-6243, from Santa Cruz Biotechnology and {alpha}-tubulin, T9026, from Sigma]. After washing the primary antibody, an appropriate secondary horseradish peroxidase-conjugated antibody was used to develop the blottings. The horseradish peroxidase chemiluminescent substrate used was enhanced chemiluminescence (Amersham) and the membranes were exposed to a blue orto CP-G film (AGFA).

Dual luciferase assays
H1299 cells were seeded in six-well plates and cultured for 24 h at 37°C. Then the medium was changed and 2 h later the cells were transiently transfected overnight with the appropriate DNAs (and additionally with phRG-TK vector, a renilla luciferase reporter) using the calcium phosphate protocol. Cells were then washed from the calcium phosphate crystals with phosphate-buffered saline and, if necessary, a glycerol shock (Dulbecco’s modified Eagle’s medium supplemented with 10% glycerol) was performed for 1 min. Fresh medium was added to the transfected cells and cultured at 37°C. p53–/–;Mdm2–/– MEFs were transfected with the appropriate DNAs, but in that case JetPEI reagent was used. Both cell types were harvested 48 h after transfection and lysed with 500 µl of 1x passive lysis buffer (Promega). The lysates were then centrifuged 30 s at 13 200 r.p.m., 4°C. Twenty microlitres of each sample was then transferred to a 96-well plate and firefly luciferase activity measured with the automated addition of 50 µl of luciferase substrate (Promega) by a Victor II (PerkinElmer, Wallac Oy) reader. Secondly, and after the automated addition of 50 µl of Stop and Glo Reagent, renilla luciferase activity was measured by the Victor II reader. Firefly luciferase activity was then normalized with renilla luciferase activity.

Quantitative reverse transcription–polymerase chain reaction
Tissue samples.
Normal tissue and tumour tissue from 20 patients with colon carcinoma, 20 patients with prostate carcinoma and 20 patients with lung carcinoma were randomly chosen from the tumour bank at the Pathology Department of Vall d’Hebrón Hospital (Barcelona, Spain). Biopsy samples were routinely collected, quickly frozen and stored at –80°C immediately after surgery. All tumours were histologically examined to confirm the diagnosis of carcinoma. All procedures of the present study have been approved by the Ethics Committee of the Vall d’Hebron Hospital.

Real-time quantitative RT–PCR.
Total RNA was extracted from normal and tumoural tissue with the RNAeasy mini kit (Qiagen, Hilden, Germany). The RNA nano Lab Chip kit (Agilent, Palo Alto, CA) was used to quantify and determine the integrity of the isolated total RNA. cDNA synthesis was done using random primers with SuperScriptTM II reverse transcriptase (Invitrogen, Carlsbad, CA) and aliquots were stored at –20°C.

Quantitative real-time TaqMan RT–polymerase chain reaction (PCR) technology (Applied Biosystems, Foster City, CA) was used to determine the differential expression of the selected genes. Relative quantification analysis was performed with the ABI PRISM 7700 instrument (Applied Biosystems). Data were analysed with sequence detection software (Applied Biosystems). The PCR cycling programme consisted of denaturing at 95°C for 10 min and 50 cycles at 95°C for 15 s and annealing and elongation at 60°C for 1 min.

The primers and TaqMan probes were purchased from Applied Biosystems.

Cyclophilin (ref. 4326316E), an endogenous control, was used to normalize variations in cDNA quantities from different samples. Each reaction was performed in triplicate with cDNA from normal and tumour tissue from each patient studied. A blank sample (no DNA) was included in each experiment. A new RNA extraction was randomly performed from the original tissue of some samples and reproducible quantitative real-time PCR results were obtained (data not shown).


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Supplementary materials
 Funding
 References
 
Generation and characterization of target cell lines
Using a retroviral vector that carries temperature-sensitive p53 (p53val135 and tsp53) to conditionally express p53 in p53–/– MEFs, we established a p53–/–;ts cell line from the infected population. Similarly, we expressed tsp53 in double p53;p21 knock out MEFs (p53–/–;p21–/–,ts). These cell lines were further infected with a retroviral vector carrying oncogenic ras (val12-ras), generating p53–/–;ts-ras and p53–/–;p21–/–;ts-ras cell lines, respectively (18). These cell lines were established and maintained at the restrictive temperature (39°C) to avoid selection against p53 or other components of the p53 pathway. Cells expressing tsp53 (p53–/–;ts, p53–/–;ts-ras, p53–/–;p21–/–;ts and p53–/–;p21–/–;ts-ras) grew at 39°C and underwent cell-cycle arrest when shifted to 32°C (Figure 1A and data not shown). Cell-cycle arrest at 32°C was more efficient in the presence of oncogenic ras. In contrast, the parental p53–/– MEFs (lacking p53val135) or expressing ras (p53–/–;ras) did not arrest at 32°C, indicating that the cell-cycle arrest we observed was not a temperature but a p53 effect (Figure 1A). p53–/–;ts-ras and p53–/–;p21–/–;ts-ras cells arrested at 32°C showed all the signs of senescence (11,18).


Figure 1
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  		Fig. 1. Growth arrest induced by p53(val135) mutant is independent of the presence of p21. (A) p53–/–;ras, p53–/–;ts and p53–/–;p21–/–;ras-ts cells were grown at restricted temperature (39°C; filled symbols) or permissive temperature (32°C; open symbols) for different days. Cells were seeded at 39°C and after 12–14 h shifted to 32°C (day 0). At indicated times, cells were fixed and their growth measured as indicated in Materials and Methods. Cellular growth was referred to cells at day 0. (open diamonds) p53–/–;ras at 32°C, (filled diamonds) p53–/–;ras at 39°C, (open squares) p53–/–;ts at 32°C, (filled squares) p53–/–;ts at 39°C, (open triangles) p53–/–;p21–/–;ras;ts at 32°C and (filled triangles) p53–/–;p21–/–;ras;ts at 39°C. Data represent the average results of one experiment in triplicate samples. Bars show standard deviations. The experiment was performed at least three times independently. (B) Scheme of the loss-of-function screening. (C) Antisense fragments against ribosomal proteins inhibit p53-induced growth arrest. In all, 6 x 104 p53–/–; ts cells were grown at restricted temperature (39°C) or permissive temperature (32°C) for 2 weeks. Then cells fixed with glutaraldehyde 0.5% and stained with 1% crystal violet to visualize colony growth. Example of the effect of two RP antisense fragments is shown.

 
Loss-of-function genetic screening
To study the genetic determinants of combined ras/p53-induced senescent-like growth arrest in the absence of p21, we have performed a screen to identify genes which loss of function bypasses the senescent phenotype in the above-described cellular system.

A random antisense fragment library generated from total polyA+ mRNA was cloned into the MMLV-based retroviral vector pMARX-IVpuro. This library contained ~6 x 106 independent clones. The library was transfected into ecotropic retrovirus-packaging cells and the replication-deficient viruses generated were then infected into exponentially growing p53–/–;p21–/–;ras;ts cells (39°C). Following selection for puromycin resistance for ~6 days, 105 cells/10 cm culture dish were plated and shifted to 32°C. After 2 weeks, plates with colonies that had overcome ras/p53-induced senescence were identified and the genomic DNA was extracted from individual clones (Figure 1B). Proviruses containing the antisense fragments were excised from the total genomic DNA and individual fragments amplified by PCR. Independent provirus carrying individual antisense fragments were retested and positive fragments identified by comparison with the BLAST database (some of them are shown in Table I).


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  		Table I. Ribosomal protein antisense fragments identified in the large-scale genetic screening

 
Among other antisense fragments, we recovered, at least twice in an independent form, antisenses against p53 mRNA. These antisenses map into a short p53 region reported previously as being efficient for mRNA antisense activity (25). These antisense fragments represent a validation of our genetic screening.

In the screening, we identified a group of antisenses against a cluster of ribosomal proteins. We recovered a total of 15 different antisense fragments from 14 different ribosomal protein genes, among them two independent antisense fragments from L6 (Table I). The size of our antisense fragments varies from 50 to 300 bp, with 14 out of 15 fragments smaller than 200 bp (Table I). This size has been reported as the most effective for gene silencing (25). The analysis of the antisense distribution along the different mRNA transcripts does not identify a specific area where the activity of the antisenses is stronger (Table I). Antisenses distribute along the entire transcripts, including the 5' non-coding sequence as in the case of the L12 antisense fragment.

Among the RPs that antisense fragments were recovered in our screening, we identified L23 and L5, two RPs reported previously to be involved in the ribosome biogenesis stress pathway connected to p53 (26–28). This fact constitutes an additional validation of our screening.

Antisense fragments against ribosomal proteins bypass p53-induced growth arrest
The different antisense fragments identified in our screening were able to bypass ras-p53-induced senescence in p21-null background. We tried to quantitate the ability of each fragment to bypass p53-induced arrest in a wild-type background. To that end, we infected the p53–/–;ts cell line with viruses carrying the different antisense fragments. Cells were selected for the presence of the provirus with a selectable marker. Then, p53–/–;ts cells expressing each of the antisense fragments were seeded at low density (6 x 104 cells/10 cm dish) in two sister plates and grown at either 32 or 39°C. After 15 days in culture, we compared the number of colonies between the plates kept at 32 or 39°C. All the antisense fragments significantly induced cellular growth at the permissive temperature (Figure 1C and Table I), bypassing the p53-induced cell-cycle arrest observed in the control plate without the antisenses. The bypassing effect ranged from 10 to 40% over the growth of the control. The different effectiveness of the antisense fragments can be attributed to many aspects, such as affinity of the antisenses for the target mRNAs or the relative weight of each ribosomal protein on the p53-dependent growth arrest. Since we analysed a pool of cells infected with similar viral title, we assume that the levels of expression of the different fragments did not vary significantly among the different cultures.

For the following experiments, we selected six of the ribosomal proteins obtained in the screening and whose relationship with this pathway had not been assessed previously.

Antisense fragments against ribosomal proteins reduce p53 transcriptional activity
p53 is a transcription factor that binds DNA and drives the transactivation of several genes that execute growth arrest (29). To assess whether the RP antisense fragments alter p53 transcriptional activity, we measured the expression of a reporter gene driven by a p53-responsive promoter ectopically co-expressed with the antisense fragments and wild-type p53. To that end, we co-transfected p53-null H1299 cells with plasmids carrying p53 cDNA under a LTR promoter, firefly luciferase under the bax promoter (p53-dependent reporter) and the antisenses under a CMV promoter. We found that almost all the antisense fragments reduced to a certain degree the p53-dependent transactivation of the bax promoter (Figure 2A), pMarX-IV empty vector did not alter the activity of p53, and the expression of MDM2 (as a positive control) reduced the transcription to basal levels. These data confirm that the majority of the different antisense fragments analysed were able to reduce the activity of p53.


Figure 2
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  		Fig. 2. Antisense fragments against ribosomal proteins inhibit p53 activity. (A) Most of the antisense fragments against ribosomal proteins inhibit p53 cDNA-induced transcriptional activation. p53-null cells H1299 were co-transfected with a reporter plasmid with the luciferase gene under bax promoter and a plasmid carrying wild-type p53 under LTR promoter. Control line one (pGL3-bax) carries the reporter construct plus an empty plasmid without p53. Lines 2–9 also carry a plasmid for the constitutive expression of the indicated antisense fragment (or controls empty vector and MDM2) under the CMV promoter. Dual luciferase activity was measured as indicated in Materials and Methods. Data represent the average results of one experiment in triplicate samples. Bars show standard deviations. The experiment was performed at least three times independently. (B) Antisense fragments against ribosomal proteins favour p53 nucleolar localization and/or degradation. HEK293 cells were transiently co-transfected with a retroviral vector encoding a chimera fusion of eGFP in the amino terminus of human p53 and retroviral vectors containing each one of the ribosomal protein antisenses. The cells were cultured and checked 48 h after transfection for the subcellular localization of eGFP-p53. The experiment was performed at least three times independently. (C) HEK293 cells were transiently co-transfected with a retroviral vector encoding a chimera fusion of eGFP in the amino terminus of human p53 and retroviral vectors containing each one of the ribosomal protein antisenses. Cells were cultured during the indicated times and checked for p53 levels by western blot. The effect of only two of the RP antisense fragments is shown. The experiment was performed at least three times independently.

 
Antisense fragments against ribosomal proteins reduce p53 levels
It has been recently shown that L11 binds to a central region in MDM2 and prevents MDM2-mediated p53 ubiquitination and degradation, subsequently restoring p53-mediated transactivation and cell-cycle arrest (30). These authors suggest that L11 functions as a negative regulator of MDM2. Therefore, if our ribosomal proteins act in a similar fashion, antisense fragments against them should produce the opposite effect. The decrease of ribosomal proteins should allow an increase of free active MDM2, thereby decreasing the levels of p53. In a different fashion, L26 has been shown to specifically enhance p53 translation through its association to regulatory sequences in the p53 mRNA in situations of biogenic stress (31). On the other hand, reduction of L26 levels triggered specific reduction of p53 mRNA translation (31), which is an MDM2-independent mechanism.

To explore the molecular mechanisms through which our antisense fragments were able to bypass p53-induced arrest, we tested first whether ectopic expression of the antisenses reduced p53 protein levels in vivo. The transfection of 293T cells with a plasmid carrying an eGFP-p53 chimera under an LTR constitutive promoter generates cells expressing GFP within the nucleus (Figure 2B). Co-transfection of the p53 chimera with MDM2 does not produce cells with homogeneously fluorescent nuclei since eGFP-p53 is targeted for degradation by MDM2. Only small condensed fluorescent dots within the nuclei were observed. This pattern of fluorescence seems to coincide with the nucleoli, and would explain the resistance to degradation. The same phenotype was observed when cells were co-transfected with the eGFP-p53 chimera and the different antisense fragments against ribosomal proteins (Figure 2B). In all the cases, GFP levels were greatly reduced, and we could find from complete abolishment of the fluorescence (as in the case of the antisenses against L5, L12 or L13) to moderate reductions (all the other cases). In most cases, we detected nucleoli-like staining, probably due to resistance to degradation once p53 is located to these bodies. The analysis by western blot of p53-GFP levels confirmed the reduction of p53 in the presence of antisense fragments against RPs (Figure 2C). Cells transfected with eGFP-p53 + MDM2 + RP antisenses showed a behaviour indistiguishable from cells transfected with eGFP–p53 + MDM2 alone (data not shown).

Finally, to discriminate between the two proposed mechanisms by which ribosomal proteins modulate the p53 protein level, we decided to express full p53 mRNA (containing the 5' and 3' untranslated region sequences) in double p53;MDM2-null cells in the presence of several of our antisense fragments. In this setting, we measured p53 levels in the absence of the negative regulation by MDM2 (Figure 3A). Our experiments showed that when p53 was transfected into cells stably expressing the antisenses against several RPs, the amount of p53 protein expressed by the cells was greatly reduced (Figure 3A). Finally, using the same cellular setting, we checked whether our antisenses against RPs were able to modulate the transcriptional activity of ectopic full-length mRNA p53 independently of MDM2. p53;MDM2-null cells stably expressing our antisense fragments were transiently co-transfected with full-length p53 and the bax-luciferase reporter. We found that all the antisense fragments analysed reduced to a certain degree the p53-dependent transactivation of the bax promoter (Figure 3B). To study whether the effect observed occurs at transcription level, we performed a dose–response experiment in the p53-null cells H1299. We transfected the cells with different amounts of plasmids carrying MDM2 or antisense fragments against S6 or S16 RPs and measured levels of protein and mRNA of p53. In these conditions, we observed a decrease in the levels of protein but no significant changes in the mRNA were observed (Figure 3C).


Figure 3
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  		Fig. 3. (A) Antisense fragments against ribosomal proteins inhibit p53 protein in MDM2-null cells. Double p53/MDM2-null MEFs were infected with retrovirus carrying the antisense fragments against S9, S16, L6, L13, S20 and L4 genes or the E6 gene. After selection, the mass culture for each RP antisense carrying population, E6 carrying population or parental cells (c) were transfected with a plasmid carrying the full p53 mRNA (Takagi et al. 2005). After 48 h, total protein was extracted and levels of p53 analysed. The experiment was performed at least three times independently. (B) All the antisense fragments against ribosomal proteins inhibit full p53 mRNA-induced transcriptional activation in MDM2-null cells. Double p53/MDM2-null MEFs were infected with retrovirus carrying the antisense fragments against S9, S16, L6, L13, S20 and L4 genes or the E6 gene. After selection, the mass culture for each RP antisense carrying population, E6 carrying population or parental cells (c) were co-transfected with a reported plasmid with the luciferase gene under bax promoter and a plasmid carrying the full p53 mRNA (Takagi et al. 2005). Line one (pGL3-bax) carries only reporter construct plus control plasmid without p53. Dual luciferase activity was measured as indicated in Materials and Methods. The experiment was performed at least three times independently. (C) p53 mRNA levels do not change with RP antisense fragments. H1299 cells were transiently co-transfected with a retroviral vector encoding a human p53 cDNA and retroviral vectors containing each one of the ribosomal protein antisenses. Cells were cultured for 48 h and checked for p53 levels by western blot (protein) and RT–PCR (full p53 mRNA). The effect of only two of the RP antisense fragments is shown.

 
These results indicate that the partial silencing of a set of RPs specifically blocks p53 translation as proposed previously for L26 (31). However, differences in response to different RP antisenses between cells with or without MDM2 suggest that other mechanisms linked to MDM2 might co-exist.

Ribosomal proteins as tumour suppressors?
Finally, if reductions in the ribosomal protein levels are able to reduce the amount of free active p53, then ribosomal proteins could behave as tumour suppressors if the cellular context is appropriate. To test this possibility, we analysed the expression of different ribosomal proteins by quantitative reverse transcription–PCR analysis comparing levels of mRNA in tumours versus mRNA in normal tissue of the same organ and patient. Samples from 20 patients of each lung, colon or prostate tumours were analysed by real-time PCR (supplementary Figure is available at Carcinogenesis Online). Results showed that ~20% of lung, prostate or colon tumours have reduced levels of S16 (Figure 4A), whereas the percentage of tumours with reduced levels of S20 is slightly lower (Figure 4A). Comparing against the number of cases showing >50% increase in specific mRNA over the non-tumoural tissue of the same patient, S16 and S20 loss in colon and lung show statistical significance (P < 0.05). Finally, only a couple of tumours (representing <5%) showed reduced levels of L6 (Figure 4A). Interestingly, in all but two cases the reduction of ribosomal protein mRNAs reaches only 50%. Finally, we compared the average amount of RP mRNA in non-tumoural samples with the average amount in tumour samples of the 20 patients in the cases where the differences were significant before. In these tumours, RP mRNA levels were significantly higher than in normal tissue (Figure 4B).


Figure 4
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  		Fig. 4. Quantitative determination of ribosomal proteins mRNA in tumour samples. We determined S16, S20 and L6 mRNA levels in three selected tumour types, including colon, lung and prostate, by quantitative PCR. mRNA from tumour and normal tissue was extracted from 20 patients of each tumour type. S16, S20 and L6 mRNA levels were quantified in each sample by Q-PCR as detailed in Materials and Methods. See supplementary Figure available at Carcinogenesis Online. (A) Data are presented as percentage of cases showing 50% of higher reduction in the mRNA levels compared with normal tissue mRNA levels in the same patient. See supplementary Figure available at Carcinogenesis Online for detailed information case by case. Statistical analysis was performed by paired t-test comparing against the percentage of samples that show >50% increase in normal samples versus tumoural samples of the same patient among the analysed population. (*) = P<0.05; (**) = P < 0.005 and (***) = P < 0.001. (B) Averaged amount of indicated ribosomal protein mRNA present in normal non-tumoural samples of the 20 patients and compared with the averaged amount present in tumour samples. Only the three statistically significant cases (A) were analysed. Statistical analysis was performed by paired t-test comparing levels of mRNA in normal samples versus tumoural samples of the same patient among the analysed population.

 

   Discussion
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 Materials and methods
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We have identified 13 different ribosomal proteins which loss of function bypasses p53-induced growth arrest. The expression of antisense fragments against these RPs reduced the transcriptional activity of p53. Our data suggest that the effect could be mediated through the control of p53 mRNA translation. However, we cannot exclude that other mechanisms linked to p53 degradation might co-exist (32–34).

Proper ribosome assembly is essential for cellular health. Therefore, it is logical that impairment of this mechanism should require a response that prevents the cell to go further growing, undergoing either cell-cycle arrest or apoptosis. Taking in consideration that most cellular stress situations are managed through p53, it is not surprising that also the response to ribosome biogenesis stress is mediated through this essential regulator. Thus, overexpression of a dominant-negative mutant of Bop1, a nucleolar protein critical for rRNA processing and ribosome assembly, inhibited 28S and 5.8S rRNA formation and led to deficiency of newly synthesized 60S ribosomal subunits in 3T3 fibroblast cells, triggering a p53-dependent G1 arrest (35). Also actinomycin D, which inhibits RNA polymerase I at a concentration of 5 nM and stalls rRNA synthesis and ribosome assembly, triggers the activation of p53 (36,37). These studies suggest the existence of a signalling pathway that senses ribosome biogenesis stress and mediates p53 activation. Indeed, ribosomal proteins L5, L11 and L23 associate with the oncoprotein MDM2 and activate p53 by inhibiting MDM2-mediated p53 ubiquitination and degradation (26–28,30,38,39). Overexpression of L5, L11 and L23 triggers p53 accumulation and activation, inducing p53-dependent G1 arrest (26–28). It has been shown that MDM2 and these ribosomal proteins can form at least two complexes in cells, MDM2–L5–L11–L23 and p53–MDM2–L5–L11–L23, and both of them seem to be independent of the polysomes (26–28). It has been proposed that the MDM2–L5–L11–L23 complex functions to inhibit MDM2-mediated p53 ubiquitination. Our data indicate that the stress caused by the partial inhibition of ribosomal protein synthesis leads to the specific inhibition of p53 mRNA translation, independently of MDM2. These results are, therefore, in accordance with those of Takagi and collaborators, which showed that partial inhibition of L26 causes a clear decrease in p53 translation (31). These authors also claim that the 5' untranslated region of p53 mRNA is essential for the regulation of p53 translation after DNA damage by ribosomal protein L26. Our data are in agreement with that hypothesis. The full-length mRNA from p53, comprising the 5' untranslated region, did not produce p53 protein in the presence of RP antisenses in MDM2-null cells (Figure 2D and E).

Recent scientific literature (33,34,40,41) suggests that while MDM2 is a key regulator of p53 function, p53 degradation can be mediated through MDM2-dependent and MDM2-independent mechanisms. MDMX activation has been reported to downregulate p53 in response to biogenesis stress (17). However, this effect seems to be also dependent on MDM2 activity. MDMX activation abrogates p53 activation and prevents growth arrest; furthermore, MDMX promotes resistance to 5-FU, which activates p53 by inducing ribosomal stress without significant DNA-damage signalling. Also, in p53 wild-type cells, ARF-BP1 directly binds and ubiquitinates p53 (33). These pathways might also be involved in the p53 regulation after downregulation of ribosomal proteins.

It is tempting to speculate that the decrease in the levels of some ribosomal proteins might contribute to tumorigenesis by decreasing p53 protein level. The analysis of tumour RNA showed a variable percentage of the samples of a variety of tumours having 50% reduction on the ribosomal protein RNA, compared with normal non-tumoural tissue from the same patient (Figure 4). However, we rarely found total loss of ribosomal protein RNA, indicating that RPs are essential, and only when other alterations co-exist, RPs' downregulation can be maintained in the tumour.

Amsterdam et al. (42) showed that many RP genes may act as haploinsufficient tumour suppressors in zebra fish. They identified 12 fish lines with elevated cancer incidence. Fish from these lines develop malignant peripheral nerve sheath tumours, and in some cases also other tumour types, with moderate to very high frequencies. Eleven of the 12 lines were heterozygous for a mutation in different ribosomal protein genes. Their findings suggest that many RP genes may act as haploinsufficient tumour suppressors in fish. The authors favour the possibility that it is a shared, ribosome-associated function that allows them to be tumour suppressors, indicating that reduced protein synthesis could lead to a reduction in the level of a critical tumour suppressor protein. Our data match with those of Amsterdam et al. and with their hypothesis.

Until now, however, no direct evidences existed about the role of RP as tumour suppressors in mammals. Only two works described this role in the bibliography. In a mouse study, two independent murine tumour cell lines were found to express tumour antigens that were indeed mutated RPs (43). In both cases, the tumours were found to become more aggressive upon either loss or mutation of the wild-type allele of the RP gene. On the other hand, in humans, ~25% of both sporadic and familial cases of Diamond–Blackfan anaemia (this syndrome involve an increased risk of leukaemia) are associated with a mutation of S19 (44). In our work, a variable percentage of tumours showed 50% reduction in the levels of RP mRNA compared with the normal tissue from the same patient. However, no 100% reduction was observed. This data would indicate that if RPs have a role as tumour suppressors, this will be in haploinsufficiency, as described previously in zebra fish (42). Although we did also find a high percentage of tumours showing an increase in RP in the tumoural tissue in other samples, according with what has been reported previously (45), it is really appealing that we did find tumours with reductions in some specific ribosomal proteins.

It is possible that ribosomal biogenesis can influence carcinogenesis in two opposite ways. On one hand, an increased metabolic rate leads to increased ribosomal biogenesis, and this would be an effect of the tumoural transformation. On the other hand, a decrease in the normal ribosomal biogenesis would be sensed as a stress, activating a checkpoint connected to p53.


   Supplementary materials
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary materials
Funding
 References
 
Supplementary Figure can be found at http://carcin.oxfordjournals.org/


   Funding
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary materials
 Funding
References
 
Fondo de Investigación Sanitaria (FIS-02/0126); Ministerio de Ciencia y Tecnología (SAF2005-00944); Fundación Mutua Madrileña and EU (from VI framework, Project COMBIO); Ministerio de Ciencia y Tecnología to M.E.C.


   Acknowledgments
 
We thank Michael Kastan for p53 full-length mRNA. The authors acknowledge the other members of the Assay Development Group at CNIO for helpful discussions and critical reading of this manuscript.

Conflict of Interest Statement: None declared.


   References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary materials
 Funding
 References
 

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Received October 10, 2007; revised December 12, 2007; accepted December 20, 2007.


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