Senescence is a mechanism that limits cellular lifespan and constitutes a barrier against cellular immortalization. To identify new senescence regulatory genes that might play a role in tumorigenesis, we have designed and performed a large-scale antisense-based genetic screen in primary mouse embryo fibroblasts (MEFs). Out of this screen, we have identified five different genes through which loss of function partially bypasses senescence. These genes belong to very different biochemical families: csn2 (component of the Cop9 signalosome), aldose reductase (a metabolic enzyme) and brf1 (subunit of the RNA polymerase II complex), S-adenosyl homocysteine hydrolase and Bub1. Inactivation, at least partial, of these genes confers resistance to both p53- and p16INK4a-induced proliferation arrest. Furthermore, such inactivation inhibits p53 but not E2F1 transcriptional activity and impairs DNA-damage-induced transcription of p21. Since the aim of the screen was to identify new regulators of tumorigenesis, we have tested their inactivation in human tumors. We have found, either by northern blot or quantitative reverse transcriptase–PCR analysis, that the expression of three genes, Csn2, Aldose reductase and Brf1, is lost at different ratios in tumors of different origins. These genes are located at common positions of loss of heterogeneity (15q21.2, 7q35 and 14q32.33); therefore,we have measured genomic losses of these specific genes in different tumors. We have found that Csn2 and Brf1 also show genomic losses of one allele in different tumors. Our data suggest that the three genes identified in the genome-wide loss-of-function genetic screen are putative tumor suppressors located at 15q21.2; 7q35 and 14q32.33.
Replicative senescence is characterized by a progressive loss of proliferative potential with the increase of population doublings, resulting in a permanent and irreversible cell-cycle arrest. Although the process of senescence occurs both in vitro and in vivo (Dimri et al., 1995; Schmitt et al., 2002; Shay and Roninson, 2004; Braig et al., 2005; Collado et al., 2005; Michaloglou et al., 2005), the transition to the senescent phenotype is commonly studied in culture where a cell population can be grown and monitored. Replicative senescence can be a consequence of shortening and dysfunction of the telomeres, due to the inability of most human cells to replicate the chromosomal ends (Serrano and Blasco, 2001; Greider and Blackburn, 2004). Nevertheless, cellular senescence (also including accelerated senescence) is a more general process not only triggered by accumulation of cell divisions, but also by activation of oncogenes (Serrano et al., 1997), DNA damage, epigenetic changes and oxidative stress (Campisi, 2001; Shay and Roninson, 2004). Senescent cells cannot be stimulated to re-enter the cell cycle by physiological mitogens, become resistant to apoptosis and acquire altered differentiated characteristics. Moreover, accelerated senescence driven by any of these stresses is independent of the telomeric length (Serrano and Blasco, 2001; Suzuki et al., 2001) and is not prevented by expression of telomerase (Morales et al., 1999). Regardless of the mechanism triggering cellular senescence, the signaling cascade leading to growth arrest seems to be similar and it involves the p53 and pRB pathways (Sherr and McCormick, 2002). Overcoming the restriction that cellular senescence poses to immortalization may be a primary step during the transformation to malignancy (Hanahan and Weinberg, 2000).
During immortalization, cells acquire genetic alterations that override the normal mechanisms controlling senescence (For review see Hanahan and Weinberg, 2000; Sherr and McCormick, 2002; Shay and Roninson, 2004; Campisi, 2005). Among the alterations that immortalize cells, those that inactivate tumor suppressor genes in the p53 and pRB pathways are very frequent in cancer. Tumor suppressor genes may get inactivated by either deletion of one or both alleles, promoter methylation, splice-site mutations and nonsense mutations that induce premature translational termination and destabilize mRNA transcripts (or a combination thereof). Such alterations result in a complete absence or partial reduction of the tumor suppressor protein in the affected cells, conferring them selective advantage in clonal selection for tumor progression.
In this work, we designed and performed a genome-wide loss-of-function genetic screen to identify additional putative tumor suppressor genes controlling senescence that might act as tumor suppressors.
Pre-senescent MEFs were infected with retrovirus carrying a library of senescent MEF's transcriptome in antisense orientation (Carnero et al., 2000). MEFs were seeded at low concentration at doubling time of 10–12 and left to senesce. Clones that were able to grow were identified, provirus recovered and sequenced.
Out of our genome-wide loss-of-function screen, we identified five different antisense fragments that targeted proteins with very different biochemical properties (Table 1). Among them, an antisense against Csn2 (a component of the Cop9 signalosome) was recovered eight independent times. Antisense fragments against Aldose reductase and S-adenosyl homocysteine hydrolase (two metabolic enzymes) and against BRF1 (a subunit of the RNA polymerase III complex) were recovered twice. Finally, an antisense against Bub1 was recovered once. The size of the fragments varies from 130 to 210 nucleotides and they map to different regions in the mRNAs, including the 3′ untranslated region, as in the case of Csn2 and Aldose reductase.
S-adenosyl homocystein hydrolase (SAHH) was identified previously in large-scale loss-of-function screen for genes involved in p53-dependent arrest and their role in cancer has been published previously in Berns et al.(2004). Suppression of SAHH by ShRNA confers resistance to both p53-dependent and p19ARF-dependent proliferation arrest; it also abolishes a DNA-damage-induced G1 cell-cycle arrest (Berns et al., 2004) and is significantly downregulated in colon and lung carcinomas (Lleonart et al., 2006).
Here, we focused on the fragments against Csn2, Arase and BRF1, since they have been related to LoH. (Supplementary Materials 1). To validate the antisense fragments, we measured their efficacy at reducing the protein levels of their respective targets. NIH3T3 cells carrying either of Csn2, ARase and BRF1 HA-tagged proteins were infected with the correspondent antisense in the pMARXIV vector, and the effect of the antisense was quantitated by western blotting. As shown in Figure 1a, the protein level of the different HA-tagged genes was reduced by average of 50%. Expression of these antisense constructs partially bypasses growth arrest in senescence and is able to produce a moderate increase in the lifespan of MEFs (Figure 1b and data not shown). In 3T3 experiments, the antisense fragments increased MEFs lifespan approximately four population doublings (Figure 1c), although the cells finally entered senescence. Moreover, the expression of the different antisense fragments also provides an early escape from senescence of the cultures. However, downregulation of these genes do not alter the p53 levels nor phosphorylation status in MEFs (Supplementary Materials 2).
In IMR90 human diploid fibroblasts, active shRNA against Csn2 and ARase do not produce viable cell lines, while active shRNA against BRF1 enhances the lifespan in 6–8 population doublings causing cells to abruptly enter apoptosis (data not shown).
Effect of antisense on pRB and p53-dependent cell growth arrest
To explore the possible mechanisms through which these genes might affect senescent arrest, we studied the effect of the different antisense fragments on the p53 and pRb checkpoints. First, we measured the ability of these antisense constructs to bypass p53-induced arrest. To this end, we expressed the different antisense fragments in a cell line derived from p53-null MEFs expressing the temperature-sensitive mutant of p53 (val135). These cells grew actively at 39 °C but arrested at 32 °C, when the p53 protein adopts the active conformation (Carnero et al., 2000). The antisense fragments inhibited the cell cycle arrest dependent on p53 with rates ranging between 20 and 40% (Figure 2a).
The antisenses affect the transcriptional activity of p53
We examined the effect of the antisense fragments on the p53-dependent transcriptional activation. A p21waf1-luciferase reporter construct was deployed as a p53 transcriptional target. All of the antisense fragments greatly reduced the transcriptional activity of p53 (Figure 2b). To check whether the p53 transcriptional activity was compromised by the antisense in a more physiological setting, we transduced NIH3T3 cells with each one of the antisense fragments, selected mass cultures carrying the retroviral vectors and analysed if DNA damage-induced p53 stabilization was followed by an increase in the p21waf1 gene expression (Figure 2c). In NIH3T3 cells as well as in the mass cultures expressing each one of the antisense fragments, p53 protein levels were increased 6 h after exposure to 0.2 μgml-1 of doxorubicin or 100 μM of cisplatin, decreasing 24 h after exposure in the case of doxorubicin and being maintained in the cisplatin-treated cells. In NIH3T3 cells, this p53 stabilization and activation produced an increase of p21waf1 protein, clearly visible 6 h after exposure to doxorubicin or cisplatin and maintained 18 h later (24 h after exposure). In the mass cultures of NIH3T3 cells expressing the antisenses, we still observed the p53 stabilization indicating that the different genes do not connect upstream of the p53 pathway. The stabilization of p53 was not translated into an increase of p21 protein, suggesting that the transcriptional activity of p53 was compromised by the antisenses (Figure 2c).
The antisense fragments help evading arrest induced by the pRB/E2F pathway
Alteration of the pRb/E2F checkpoint has also been described to alter the replicative senescence in MEFs (Yamasaki et al., 1998; Sage et al., 2000). Therefore, we have determined whether the overexpression of the different antisense fragments has any effect on E2F-induced transactivation. To that end, we co-transfected HEK293 cells with human E2F, a synthetic E2F promoter-luciferase reporter, and each one of the antisense fragments (Figure 2d). None of the antisense fragments significantly affected the expression of luciferase, indicating that the proteins they shut down do not interfere with the transcriptional activity of E2F1. Then, we investigated whether the antisense fragments were able to bypass p16-induced cell cycle arrest by performing a colony formation assay. We first transduced wild-type MEFs with the antisense fragments and selected mass cultures, expressing them with the appropriate antibiotic. Then, each mass culture was transduced with p16INK4a at low viral titer and cultured for 2 weeks under the appropriate culture selection. Cells rapidly entered growth arrest, as can be seen in the control plate (Figure 2e), but in those plates where each one of the antisenses were expressed, many cells escaped arrest and formed colonies. In this case, the most effective antisense was against Aldose reductase.
Human tumors frequently lose the expression of the candidate tumor suppressor genes
We have shown that Csn2, Aldose reductase and BRF1 protein loss-of-function can partially extend MEF lifespan, probably by regulating p53 transcription and by controlling p16ink4a-induced arrest, supporting a molecular mechanism for their biological role in senescence. Since loss of function might regulate the effectors of senescence and the p53 and pRB pathways, we have directly analysed the involvement of these genes in human tumors.
First, we checked the expression of the different genes of interest in several arrays of paired normal/tumoral tissue samples from the same patients. cDNA probes from each one of the candidate genes were radioactively labeled and hybridized with the array of tumoral/normal RNA samples. As an expression control, we hybridized the arrays with an ubiquitin-specific probe. We normalized the signals of the specific probes against the signal of ubiquitin and quantitated the signal in tumor and normal samples. Finally, we identified those tumor samples in which the signal was decreased by at least 50% in tumors with respect to normal tissue (Figure 3a). We find that all three of our putative tumor suppressor genes had a marked reduction of their expression in some tumoral samples. An especially high percentage of the tumoral samples showed at least a 50% reduction of the expression of Csn2 in thyroid tumors (Figure 3a). High percentages of Csn2 mRNA loss were observed in tumors of pancreas, breast, ovary, kidney, uterus and rectum. BRF1 mRNA was at least 50% lost in some ovary, kidney, uterus and rectum tumors (Figure 3a). Aldose reductase was lost in a few tumor types, mainly in thyroid, uterus, lung and stomach and at a lower percentage than the other two genes (Figure 3a).
To complement these studies, we have performed quantitative analysis of the expression of Csn2, ARase and BRF1 in three different types of tumors: lung, colon and prostate. In each case, we quantified the mRNA in the tumoral sample versus the normal sample of the same tissue of the same patient (Figure 3b and Supplementary Materials 3). Our data show that all of the genes are reduced by at least 50% in the three types of tumors (Figure 3b). When we averaged the total level of mRNAs of the different genes in normal tissue and compared these levels with the same in the tumoral samples, we found a consistent reduction of the Csn2, ARase and BRF1 mRNAs in the tumoral samples (Figure 3c).
Candidate tumor suppressor genes are located in chromosomal regions frequently lost in cancer
Finally, we wanted to investigate whether the genes identified in our loss-of-function screening were affected by allelic loss in cancer, since they are located in chromosomal regions significantly lost (>20%) in different tumors, according to data obtained in a CGH tumor database (see Supplementary Information 3). Thus, Csn2 is located at 15q21.2, Aldose reductase at 7q35, and BRF1 at 14q32.33. Therefore, we have explored whether these genes could be the putative tumor suppressors localized in these loci.
To that end, we analysed the allelic losses of these genes in DNA from tumor samples of different origins. We analysed samples from thyroid (n=31), kidney (n=9, all clear-cell renal carcinoma), larynx (n=16), colon (n=13) and rectum (n=12) tumors (Ref 4). Analysis of Csn2 and BRF1 showed single allelic genomic losses in all tumor types analysed (Figure 4a) with variations amongst tumor types. The analysis of Aldose Reductase was hampered by the existence of several pseudogenes that complicated the interpretation of the data (data not shown).
Csn2 showed specific allelic losses in 38.7% of thyroid tumors correlating with the high levels of mRNA losses detected previously in Figure 3a. Cns2 was also lost in 33.3% of clear cell renal carcinomas, 6.25% of larynx tumors, 23% rectum and 30% in colon carcinomas (Figure 4b). Also HBRF1 was lost in 50% CCRC and in 23% of rectal tumors (Figure 4a). Nevertheless, the percentage of HBRF1 loss in other tumors was smaller, accounting for 6.25% in larynx, 7% in thyroid and 7.7% in colon.
Csn2, BRF1 and ARase reduced the foci induced by oncogenic ras
To finally assess the capability of these genes to act as tumor suppressors we analysed the alteration of the oncogenic susceptibility induced in the absence or presence of the genes identified. To such extent we expressed oncogenic ras in NIH3T3 cells previously expressing each of the antisense fragment identified. After selection, cells were cultured in soft agar and the ability to grow in semisolid media assessed. We observed that the different antisenses increased the growth potential in soft agar in around 50% (Figure 5a). A further prerequisite to be considered as tumor suppressor candidate is the ability of the genes to reduce the oncogenic potential. To that end, we coexpressed oncogenic ras and either of the full-length cDNAs of the genes identified in a classical foci formation assay (Carnero and Beach, 2004). Ras induced a number of foci in NIH3T3 (Figure 5b). However, the ectopic overexpression of Csn2, BRF1 or Arase almost completely abolish foci formation (Figure 5b). The fact that the absence of the genes increases transforming properties and its overexpression reduced these properties indicate that these genes might be potential tumor suppressor candidates.
Exploiting the entry of MEFs into culture stress-induced senescence, we have identified three genes through which loss of function, at least partially, might downregulate growth arrest. These three genes belong to very different biochemical families and have not been associated before with this phenotype. Furthermore, the analysis of the genetic status of these genes shows that they are lost through different mechanisms in tumor samples from several tissues. These results suggest that these genes might function as tumor suppressors and their loss could confer some advantage to tumoral cells.
Csn2 is a subunit of the COP9 signalosome, a multimeric protein complex conserved among most eukaryotes (Wei et al., 1998; Bech-Otschir et al., 2002). Disruption of csn2 in mice produced p53 accumulation and early embryonic death (Lykke-Andersen et al., 2003). It was shown that COP9 signalosome-associated kinase activity phosphorylates p53 in the core domain and targets it for proteasomal degradation (Bech-Otschir et al., 2001). Recently, it has been proposed that the Cop9 signalosome is essential for the activity of MLF1, a negative regulator of cell-cycle progression functioning upstream the tumor suppressor p53 (Yoneda-Kato et al., 2005). Disruption of Csn2 by anti-Csn2 antibodies inhibits signalosome-dependent p27 stabilization and G1/S arrest (Yang et al., 2002). Knockdown of the Csn3 protein by siRNA interferes with the activation of p53 by genotoxic stress, implying that the COP9 signalosome is an essential factor in the chain of events leading to p53 activation after DNA damage (Yoneda-Kato et al., 2005). Our data support the notion that the COP9 signalosome is essential for p53 activity, since depletion of csn2 with antisense noticeably affects the ability of p53 to induce cell cycle arrest. Furthermore, the same antisense partially abolishes p53 transactivation of the p21 promoter and impairs the increase of p21 protein after treatment with DNA damaging agents, indicating that the COP9 signalosome acts somehow downstream p53.
BRF1 is a subunit of the RNA polymerase III transcription factor IIIB. RNA polymerase III synthesizes several small RNA species, such as the tRNAs and 5 S rRNA. In cell cycle-arrested cells, BRF1 levels are markedly reduced due to a decrease in protein stability (Eichhorn and Jackson, 2001). Furthermore, pRB regulates RNA polymerase III somehow binding to BRF1 (Larminie et al., 1997). We do not have any mechanistic insight about the mode of action of Brf1 on the p53 pathway impairing p21 transcription.
Aldose reductase (220.127.116.11) is an enzyme that participates in glucose metabolism, plays a protective role against toxic aldehydes and is important for osmoregulation in the kidney. There is some evidence that expression of AKR1B1 is decreased in adrenocortical cancer (Lefrancois-Martinez et al., 2004).
The expression of the three genes obtained in our screening was altered in tumoral tissue when compared with matched non-tumoral samples from the same tissue and patient. We observed that Csn2 expression was reduced in 18.5% of the tumors analysed, with 50% of thyroid tumors showing complete disappearance (5 of 10 samples analysed). The expression of the other two genes was lost with lower frequencies, indicating that their activity can probably be lost through other mechanisms, such as mutations or post-translational modifications. We did not investigate whether each one of the genes behave as tumor suppressors in a specific tissue, but the high specificity of the loss of expression of each gene in very unique tumoral tissues strongly suggests this possibility.
The three genes identified in our loss-of-function screening are located in chromosome regions with significant (>20%) allelic loss in different tumors. Csn2 is located at 15q21.2, a region frequently lost in a variety of tumors of different origins and with a higher incidence in lung, breast, brain and colorectal carcinomas. The average incidence of csn2 allelic loss across different tumors (26.99%) is in the range observed for well known tumor suppressor genes, such as pRB (38.64%), PTEN (30.68%) or BRCA2 (24.01%). This observation is in agreement with the existence of a candidate tumor suppressor gene in the 15q21.2 locus. Our hypothesis is further supported with detailed cytogenetic studies using microsatellite probes to detect specific loss of heterogeneity Frequency of loss ranges from 27% in cervical carcinomas to 62% in head and neck squamous cell carcinomas (HNSCC) (See Table 2 in Supplementary Information). The aldose reductase gene is located at 7q35. This region has a lower incidence of loss in tumors, still being significant in some tumors such as HNSCC (22.22%) (See Table 1 in Supplementary Information). LOH analysis shows a higher impact, especially in oropharyngeal epithelial carcinoma, with a 50% allelic loss (see Table 2, in Supplementary Information). A similar situation accounts for BRF1, at 14q32.33. CGH analysis shows a moderate loss, with a higher incidence (20.69%) in colorectal carcinomas. LOH data confirms this observation, providing higher values of allelic loss (up to 66% in metastatic renal carcinoma) and affecting a variety of tumor types (See Supplementary Information).
The data presented in this paper support the notion that the genes obtained in the screening could participate as tumor suppressors in the cancer-safeguarding mechanism of senescence. Further research will be necessary to fully understand the mechanism of action of all three genes, and to add new insight into the possible prognostic value of the loss or mutation of these genes in cancer.
Materials and methods
MEFs were generated from 13.5 day CD1 mouse embryos. The head and blood organs were eliminated and the rest of the embryo was minced and dispersed in 0.1% trypsin (60 min at 37 °C). Adherent cells growing in Dulbecco's modified Eagle's medium (Gibco, Invitrogen SA, Barcelona, Spain) supplemented with 10% FBS (Sigma) were cultured during 2 population doublings and then frozen. MEFs were subcultured 1:4 when they reached confluence; each passage was considered two population doublings (PDs). For colony-formation assays, cells were plated at 103 cells per 10 cm plate. After 10 days, colonies were stained with crystal violet and counted.
3T3 protocol. Every 3 days, cells were trypsinized, counted and 106 cells were plated per 10-cm plate. The relative number of cells is considered as a measure of the number of cells per passage related to the initial number of cells seeded per plate.
Growth in soft agar
To measure the anchorage-independent growth, 104 cells were suspended in 1.4% agarose D-1 Low EEO (Pronadisa) growth medium containing 10% FBS, disposed onto a solidified base of growth medium containing 2.8%. agar (agarose D-1 Low EEO, Pronadisa), and overlaid with 1 ml of growth medium. After 24 h, media containing 10% FBS were added to each 35 mm dish and renewed two times weekly. Colonies were scored 3 weeks after, and all values were determined in triplicate.
In all 50% confluent NIH3T3cells were transfected with 0.1 ìg of pbabepuro-ras (val12) and 0.5 ìg of pwzl-hygro alone or carrying the full length cDNA of Csn2, Arase or BRH1 using Jet-Pei as indicated by the vendor. Cells were cultured 15 days, then fixed and stained with crystal violet.
Library generation, transduction and recovery of the active proviruses
As a source of the mRNA, we used 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, selected by size (50–500 nucleotides) on a S400 column (Pharmacia and Upjohn SA, Barcelona, Spain) and cloned into the EcoRI and XhoI sites of pMARXIVpuro in the antisense orientation.
Transduction of the library and recovery of the proviruses was performed as described previously by Carnero and co-workers (Carnero et al., 2000).
Western blot analysis
Cells were washed twice with ice-cold phosphate-buffered saline (PBS) and lysed by sonication in lysis -buffer (50 mM Tris–HCl pH 7.5, 1% NP-40, 10% glycerol, 150 mM NaCl, 2 mM Complete protease inhibitor cocktail Roche Diagnostics, Barcelona, Spain). The protein content of the lysates was determined by the modified method of Bradford using bovine serum albumin (BSA) as a standard. The appropriate quantity of protein was then dissolved in Laemmli buffer (62.5 mM Tris–HCl pH 6.8, 10% (v/v) glycerol, 1% (w/v) SDS, 5% (w/v) 2-mercaptoethanol, 0.0025% (w/v) bromphenol blue -Sigma-) and these samples were then separated by SDS–PAGE, transferred on to Immobilon-P membrane (Millipore, Iberica SA, Madrid, Spain), and immunostained. The following primary antibodies were used: PAb FL-393 anti-p53 (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA; sc-6243), MAb anti-p21 (Oncogene Research Products, EMD Biosciences Inc., San Diego, CA, USA; OP64), MAb anti-HA (Sigma-Aldrich, St Louis, MO, USA; clone HA7), MAb anti-α tubulin (Sigma T9026), Ab anti-p53 P-ser18 (Cell Signalling Technology, Danvers, MA, USA), and horseradish peroxidase-labeled rabbit anti-mouse (Promega Corp., Madison, WI, USA; 17331001), goat anti-rabbit (Calbiochem, EMD Biosciences Inc., San Diego, CA, USA; 401315) and rabbit anti-goat (Santa Cruz sc-2768) as secondary antibodies. Proteins were visualized using the ECL detection system (Amersham Biosciences, Fairfield, CT, USA).
RNA array hybridization
Cancer profiling array membranes (BD Biosciences, Franklin Lanes, NJ, USA) were pre-hybridized with ExpressHyb hybridization solution for 4 h at 65 °C. The appropriate probe was then labeled by PCR with 50 °C of redivue dCTP32 (Amersham), using the primers previously described for the reverse transcriptase–PCR. The labeled probe was then purified from free hot nucleotides with a sepharose G-50 column Nick (Amersham). The purified probe was then denatured for 3 min at 100 °C and added to the hybridization solution. The hybridization was performed overnight at 65 °C. Then the membrane was washed at 65 °C two times with 2 × SSPE, 0.1% SDS; once with 1 × SSPE, 0.1% SDS and once more with 0.1 × SSPE, 0.1% SDS. The membrane was then exposed to a Biomax MS film (Kodak).
Normal tissue and tumor tissue from 20 patients with colon carcinoma, 20 patients with prostate carcinoma and 20 patients with lung carcinoma were randomly chosen from the tumor bank at the Pathology Department of Vall d'Hebrón Hospital (Barcelona, Spain). Biopsy samples are routinely collected, quickly frozen and stored at –80°C immediately after surgery. All tumors were histologically examined to confirm the diagnosis of carcinoma. All procedures of the present study have been approved by the Ethics Committee of the Hospital Vall d'Hebron.
Total RNA was extracted from normal and tumor tissue with the RNAeasy mini kit (Qiagen, Hilden, Germany). The RNA nano Lab Chip kit (Agilent, Palo Alto, CA, USA) 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, USA) and aliquots were stored at −20 °C.
Quantitative real-time TaqMan reverse transcriptase–PCR technology (Applied Biosystems, Foster City, CA, USA) 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. The PCR cycling program 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.
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 tumor 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).
Multiplexed genomic loss detection
To investigate the presence of deletions affecting the BRF1 and Csn2 genes, we designed a technique based on specific multiplex amplification of each gene. We first designed and labeled (5′ 6-FAM) a pair of primers for each exon. The primer pairs for amplification were designed on the basis of genomic sequences of BRF1 and Csn2 (Entrez Gene ID: 2972 and 9318, respectively), and were as follows: TRIP15-1F (5′-IndexTermAGCTGAGAGTGACGCCTCTG-3′); TRIP15-1R (5′-IndexTermATCACCACCCTCAGAGTTCC-3′); TRIP15-6F (5′-IndexTermCAGGTACAAATTTGCTGTTTACAA-3′); TRIP15-6R (5′-IndexTermAGCTACCTGGCACGACTGAT-3′); TRIP15-10F (5′-IndexTermTTGAGAAGCAGGGTCTTTGG-3′); TRIP15-10R (5′-IndexTermTGGCATTTCTTTGACAGTGG-3′); TRIP15-13F (5′-IndexTermTTTAATGTAGGCAGCTGCTTTTT-3′); TRIP15-13R (5′-IndexTermGCACCACCCCTCTTCTGAT-3′); BRF1-2F (5′-IndexTermCCCGCTTTGTTCTCACTTTG-3′); BRF1-2R (5′-IndexTermCGGTGGGAAATACCATTCTG-3′); BRF-5F (5′-IndexTermACCAATGCTGTTTCTGGTTG-3′); BRF-5R (5′-IndexTermGCACGCTGGTCCCTACAG-3′); BRF-8F (5′-IndexTermAGCTCCATTTCCCATGTCTG-3′); BRF-8R (5′-IndexTermGCTCTGCTACAACCCCAGTC-3′); BRF-17F (5′-IndexTermGGTCTGAGGGGTCTGTTGAG-3′); BRF-17R (5′-IndexTermTGAAGCTAACAGGCCCAAGT-3′). We used control fragments from chromosomes 1, 3 and 11 as internal controls of the assay. We amplified genomic DNA of tumors by means of a multiplex PCR kit, following standard recommendations (Qiagen GmbH). Briefly, multiplex amplifications were performed in 25 μl of a mixture containing 1 × multiplex PCR master mix, 0.2 μM of each primer, and 100–200 ng of genomic DNA. The PCR program started with an initial heat-activating step at 95 °C for 15 min to activate HotStartTaq DNA polymerase. Amplification was for 20 cycles: 30 s at 94 °C, 90 s at 60 °C and 90 s at 72 °C, followed by 10 min of final extension at 72°C. PCR amplification products obtained from the deletion tests were used for fragment analysis on an ABI PRISMTM 310 capillary sequencer (Applied Biosystems, Perkin Elmer), and analysed using GeneScan v3.1 (Applied Biosystems). Normalization was performed by overlapping tumor samples with a control sample, determining the peak surface of all fragments and calculating the normal peak fractions (Schouten et al., 2002).
Cells were seeded in 6-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. The cells were then washed from the calcium phosphate crystals with PBS and a glycerol shock (DMEM supplemented with 10% glycerol) was performed during 1 min. Fresh medium was added to the transfected cells and cultured at 37 °C. Cells were harvested 48 h after transfection and lysed with 200 μl of 1 × Reporter Lysis Buffer (Promega). The lysates where then frozen, thawed and centrifuged 2 min at 13 200 r p m , 4 °C. Twenty microliters of each sample were then transferred to a 96 -well plate and luciferase activity measured with the automated addition of 100 ml of luciferase substrate (Promega) by a Victor II (Wallac Oy) reader. Firefly luciferase activity was then normalized with Renilla luciferase activity.
Statistical analysis was performed by unpaired t- test comparing against the percentage of samples that show >50% increase in normal samples vs tumoral samples of the same patient (Figure 3), against the frequency of allelic loss in non tumoral samples (Figure 4). *P<0.05; **P<0.005; ***P<0.001.
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We thank the CNIO Tumor Bank for providing the tumor samples. This work has been partly supported by grants from Spanish Ministry of Health (FIS-02/0126), Fundación Mutua Madrileña and the Spanish Ministry of Education and Science (SAF2005-00944) to AC and MRC and Wellcome Trust (to DHB).
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Leal, J., Fominaya, J., Cascón, A. et al. Cellular senescence bypass screen identifies new putative tumor suppressor genes. Oncogene 27, 1961–1970 (2008). https://doi.org/10.1038/sj.onc.1210846
- genetic screening
- tumor suppressor
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