Tissue Microarray Cytometry Reveals Positive Impact of Homeodomain Interacting Protein Kinase 2 in Colon Cancer Survival Irrespective of p53 Function
2011; Elsevier BV; Volume: 178; Issue: 5 Linguagem: Inglês
10.1016/j.ajpath.2011.01.021
ISSN1525-2191
AutoresIsabelle Soubeyran, Isabelle Mahouche, Aude Grigoletto, Thierry Lesté-Lasserre, Guillaume Drutel, Christophe Rey, S. Pédeboscq, France Blanchard, Véronique Brouste, Jean‐Christophe Sabourin, Y. Bécouarn, Josy Reiffers, François Ichas, Francesca De Giorgi,
Tópico(s)Hedgehog Signaling Pathway Studies
ResumoThe human p53 gene is a tumor suppressor mutated in half of colon cancers. Although p53 function appears important for proliferation arrest and apoptosis induced by cancer therapeutics, the prognostic significance of p53 mutations remains elusive. This suggests that p53 function is modulated at a posttranslational level and that dysfunctions affecting its modulators can have a prognostic impact. Among p53 modulators, homeodomain interacting protein kinase (HIPK) 2 emerges as a candidate “switch” governing p53 transition from a cytostatic to a proapoptotic function. Thus, we investigated the possible prognostic role of HIPK2 on a retrospective series of 80 colon cancer cases by setting up a multiplexed cytometric approach capable of exploring correlative protein expression at the single tumor cell level on TMA. Crossing the data with quantitative PCR and p53 gene sequencing and p53 functional assays, we observed the following: despite a strong impact on p21 transcription, the presence of disabling p53 mutations has no prognostic value, and the increased expression of the HIPK2 protein in tumor cells compared with paired normal tissue cells has a strong impact on survival. Unexpectedly, HIPK2 effect does not appear to be mediated by p53 function because it is also observed in p53-disabling mutated backgrounds. Thus, our results point to a prominent and p53-independent role of HIPK2 in colon cancer survival. The human p53 gene is a tumor suppressor mutated in half of colon cancers. Although p53 function appears important for proliferation arrest and apoptosis induced by cancer therapeutics, the prognostic significance of p53 mutations remains elusive. This suggests that p53 function is modulated at a posttranslational level and that dysfunctions affecting its modulators can have a prognostic impact. Among p53 modulators, homeodomain interacting protein kinase (HIPK) 2 emerges as a candidate “switch” governing p53 transition from a cytostatic to a proapoptotic function. Thus, we investigated the possible prognostic role of HIPK2 on a retrospective series of 80 colon cancer cases by setting up a multiplexed cytometric approach capable of exploring correlative protein expression at the single tumor cell level on TMA. Crossing the data with quantitative PCR and p53 gene sequencing and p53 functional assays, we observed the following: despite a strong impact on p21 transcription, the presence of disabling p53 mutations has no prognostic value, and the increased expression of the HIPK2 protein in tumor cells compared with paired normal tissue cells has a strong impact on survival. Unexpectedly, HIPK2 effect does not appear to be mediated by p53 function because it is also observed in p53-disabling mutated backgrounds. Thus, our results point to a prominent and p53-independent role of HIPK2 in colon cancer survival. The human p53 gene acts as a tumor suppressor that plays a central role in protecting the genome against genotoxic stresses, such as oxidative stress, UV light, and ionizing radiation. In such conditions, p53 “senses” the DNA lesions and is activated, leading to the transactivation of target genes involved in cell cycle arrest and apoptosis. This either allows for DNA repair or, alternatively, when the damage is extensive, provokes cell self-elimination by apoptosis.1Vousden K.H. Prives C. Blinded by the light: the growing complexity of p53.Cell. 2009; 137: 413-431Abstract Full Text Full Text PDF PubMed Scopus (2315) Google Scholar Although tumor protein 53 (TP53) is mutated in approximately 50% of colon cancer cases,2Iacopetta B. TP53 mutation in colorectal cancer.Hum Mutat. 2003; 21: 271-276Crossref PubMed Scopus (253) Google Scholar the prognostic significance of these mutations remains controversial. In a large cohort of colorectal carcinomas published by the TP53-CRC Collaborative Study Group, this prognostic impact seems intricate and depends on tumor site, type of mutation, adjuvant therapy, and stage of the disease.3Iacopetta B. Russo A. Bazan V. Dardanoni G. Gebbia N. Soussi T. Kerr D. Elsaleh H. Soong R. Kandioler D. Janschek E. Kappel S. Lung M. Leung C.S. Ko J.M. Yuen S. Ho J. Leung S.Y. Crapez E. Duffour J. Ychou M. Leahy D.T. O'Donoghue D.P. Agnese V. Cascio S. Di Fede G. Chieco-Bianchi L. Bertorelle R. Belluco C. Giaretti W. Castagnola P. Ricevuto E. Ficorella C. Bosari S. Arizzi C.D. Miyaki M. Onda M. Kampman E. Diergaarde B. Royds J. Lothe R.A. Diep C.B. Meling G.I. Ostrowski J. Trzeciak L. Guzinska-Ustymowicz K. Zalewski B. CapellÁ G.M. Moreno V. Peinado M.A. Lönnroth C. Lundholm K. Sun X.F. Jansson A. Bouzourene H. Hsieh L.L. Tang R. Smith D.R. Allen-Mersh T.G. Khan Z.A. Shorthouse A.J. Silverman M.L. Kato S. Ishioka C. TP53-CRC Collaborative GroupFunctional categories of TP53 mutation in colorectal cancer: results of an International Collaborative Study.Ann Oncol. 2006; 17: 842-847Crossref PubMed Scopus (84) Google Scholar, 4Russo A. Bazan V. Iacopetta B. Kerr D. Soussi T. Gebbia N. The TP53 colorectal cancer international collaborative study on the prognostic and predictive significance of p53 mutation: influence of tumor site, type of mutation, and adjuvant treatment.J Clin Oncol. 2005; 23: 7518-7528Crossref PubMed Scopus (312) Google Scholar Posttranslational modifications of the p53 protein, such as ubiquitination, phosphorylation, and acetylation, contribute to p53 activity regulation, leading to protein stabilization, conformational changes, and modifications of its affinity for DNA and its interaction with transcriptional coactivator complexes.5Kruse J.P. Gu W. Modes of p53 regulation.Cell. 2009; 137: 609-622Abstract Full Text Full Text PDF PubMed Scopus (1283) Google Scholar As a result, the molecular modulators of p53 could play a significant role by influencing the p53-dependent responses in tumor cells and altering the prognostic significance of the p53 genetic status of the tumor. Among such modulators, homeodomain interacting protein kinase 2 (HIPK2)6Kim Y.H. Choi C.Y. Lee S.J. Conti M.A. Kim Y. Homeodomain-interacting protein kinases, a novel family of co-repressors for homeodomain transcription factors.J Biol Chem. 1998; 273: 25875-25879Crossref PubMed Scopus (249) Google Scholar seems to be an important regulator of p53 functions acting through direct phosphorylation of p53 at Ser46 in response to severe DNA damage.7D'Orazi G. Cecchinelli B. Bruno T. Manni I. Higashimoto Y. Saito S. Gostissa M. Coen S. Marchetti A. Del Sal G. Piaggio G. Fanciulli M. Appella E. Soddu S. Homeodomain-interacting protein kinase-2 phosphorylates p53 at Ser 46 and mediates apoptosis.Nat Cell Biol. 2002; 4: 11-19Crossref PubMed Scopus (574) Google Scholar, 8Dauth I. Kruger J. Hofmann T.G. Homeodomain-interacting protein kinase 2 is the ionizing radiation-activated p53 serine 46 kinase and is regulated by ATM.Cancer Res. 2007; 67: 2274-2279Crossref PubMed Scopus (67) Google Scholar, 9Hofmann T.G. Möller A. Sirma H. Zentgraf H. Taya Y. Dröge W. Will H. Schmitz M.L. Regulation of p53 activity by its interaction with homeodomain-interacting protein kinase-2.Nat Cell Biol. 2002; 4: 1-10Crossref PubMed Scopus (499) Google Scholar The phosphorylation of p53 Ser46 may represent a sensor of DNA damage intensity that promotes a shift in p53 affinity from cell cycle arrest–related promoter genes to apoptosis genes.10Mayo L.D. Seo Y.R. Jackson M.W. Smith M.L. Rivera Guzman J. Korgaonkar C.K. Donner D.B. Phosphorylation of human p53 at serine 46 determines promoter selection and whether apoptosis is attenuated or amplified.J Biol Chem. 2005; 280: 25953-25959Crossref PubMed Scopus (126) Google Scholar, 11Oda K. Arakawa H. Tanaka T. Matsuda K. Tanikawa C. Mori T. Nishimori H. Tamai K. Tokino T. Nakamura Y. Taya Y. p53AIP1, a potential mediator of p53-dependent apoptosis, and its regulation by Ser-46-phosphorylated p53.Cell. 2000; 102: 849-862Abstract Full Text Full Text PDF PubMed Scopus (1024) Google Scholar, 12Rinaldo C. Prodosmo A. Mancini F. Iacovelli S. Sacchi A. Moretti F. Soddu S. MDM2-regulated degradation of HIPK2 prevents p53Ser46 phosphorylation and DNA damage-induced apoptosis.Mol Cell. 2007; 25: 739-750Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar HIPK2 also forms a heterocomplex with p53, acetyltransferase Creb-binding protein, and promyelocytic leukemia protein in promyelocytic leukemia protein–nuclear bodies; and the phosphorylation of p53 at Ser46 by HIPK2 promotes the acetylation of p53 by acetyltransferase Creb-binding protein at lysines 373/382, playing a critical role in the induction of apoptosis.9Hofmann T.G. Möller A. Sirma H. Zentgraf H. Taya Y. Dröge W. Will H. Schmitz M.L. Regulation of p53 activity by its interaction with homeodomain-interacting protein kinase-2.Nat Cell Biol. 2002; 4: 1-10Crossref PubMed Scopus (499) Google Scholar, 13Guo A. Salomoni P. Luo J. Shih A. Zhong S. Gu W. Pandolfi P.P. The function of PML in p53-dependent apoptosis.Nat Cell Biol. 2000; 2: 730-736Crossref PubMed Scopus (387) Google Scholar, 14Pearson M. Carbone R. Sebastiani C. Cioce M. Fagioli M. Saito S. Higashimoto Y. Appella E. Minucci S. Pandolfi P.P. Pelicci P.G. PML regulates p53 acetylation and premature senescence induced by oncogenic Ras.Nature. 2000; 406: 207-210Crossref PubMed Scopus (1130) Google Scholar Moreover, the main mechanism of p53 expression-level regulation that is mediated by the ubiquitin ligase MDM2 is controlled by HIPK2. Indeed, HIPK2 inhibits the nuclear export and ubiquitination of p53 mediated by MDM2, thus neutralizing p53 degradation and promoting p53-dependent apoptosis.15Di Stefano V. Blandino G. Sacchi A. Soddu S. D'Orazi G. HIPK2 neutralizes MDM2 inhibition rescuing p53 transcriptional activity and apoptotic function.Oncogene. 2004; 23: 5185-5192Crossref PubMed Scopus (61) Google Scholar In addition, the phosphorylation of p53 at Ser46 by HIPK2 inhibits the MDM2 negative feedback loop by inhibiting p53-mediated MDM2 gene transcription. In addition, HIPK2 further inhibits MDM2 in a p53-independent and transcription-independent way: MDM2 can be phosphorylated by HIPK2, causing its cytoplasmic shuttling and provoking its proteasomal degradation.16Di Stefano V. Mattiussi M. Sacchi A. D'Orazi G. HIPK2 inhibits both MDM2 gene and protein by, respectively, p53-dependent and independent regulations.FEBS Lett. 2005; 579: 5473-5480Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar From a functional point of view, the knockdown of HIPK2 abolished repair after DNA damage in vitro.17Nardinocchi L. Puca R. Sacchi A. D'Orazi G. HIPK2 knock-down compromises tumor cell efficiency to repair damaged DNA.Biochem Biophys Res Commun. 2007; 361: 249-255Crossref PubMed Scopus (8) Google Scholar Moreover, in a recent study,18Puca R. Nardinocchi L. Gal H. Rechavi G. Amariglio N. Domany E. Notterman D.A. Scarsella M. Leonetti C. Sacchi A. Blandino G. Givol D. D'Orazi G. Reversible dysfunction of wild-type p53 following homeodomain-interacting protein kinase-2 knockdown.Cancer Res. 2008; 68: 3707-3714Crossref PubMed Scopus (63) Google Scholar a significant correlation between poor survival rates and low mRNA HIPK2 expression was reported in wild-type (WT) p53 colonic tumors. HIPK2 can also modulate the apoptotic response in the absence of p53. Indeed, HIPK2 facilitates the phosphorylation of the antiapoptotic corepressor C-terminal binding protein at Ser422, directly and indirectly, through the c-Jun N-terminal kinase signaling pathway, resulting in C-terminal binding protein proteasomal degradation and the promotion of apoptosis.19Hofmann T.G. Stollberg N. Schmitz M.L. Will H. HIPK2 regulates transforming growth factor-beta-induced c-Jun NH(2)-terminal kinase activation and apoptosis in human hepatoma cells.Cancer Res. 2003; 63: 8271-8277PubMed Google Scholar, 20Wang S.Y. Iordanov M. Zhang Q. c-Jun NH2-terminal kinase promotes apoptosis by down-regulating the transcriptional co-repressor CtBP.J Biol Chem. 2006; 281: 34810-34815Crossref PubMed Scopus (45) Google Scholar, 21Zhang Q. Nottke A. Goodman R.H. Homeodomain-interacting protein kinase-2 mediates CtBP phosphorylation and degradation in UV-triggered apoptosis.Proc Natl Acad Sci U S A. 2005; 102: 2802-2807Crossref PubMed Scopus (70) Google Scholar Furthermore, HIPK2 appears to be highly regulated at both the activity and expression levels.22Sombroek D. Hofmann T.G. How cells switch HIPK2 on and off.Cell Death Differ. 2009; 16: 187-194Crossref PubMed Scopus (57) Google Scholar For instance, in response to severe DNA damage, caspase-6, a p53 target gene, removes an inhibitory C-terminal domain from HIPK2, resulting in a hyperactive kinase that potentiates p53 Ser46 phosphorylation.23Calzado M.A. Renner F. Roscic A. Schmitz M.L. HIPK2: a versatile switchboard regulating the transcription machinery and cell death.Cell Cycle. 2007; 6: 139-143Crossref PubMed Scopus (108) Google Scholar, 24Gresko E. Roscic A. Ritterhoff S. Vichalkovski A. del Sal G. Schmitz M.L. Autoregulatory control of the p53 response by caspase-mediated processing of HIPK2.EMBO J. 2006; 25: 1883-1894Crossref PubMed Scopus (66) Google Scholar Regarding expression, MDM2 was reported to act as a negative regulator of HIPK2, mediating its ubiquitination and degradation on mild DNA damage.12Rinaldo C. Prodosmo A. Mancini F. Iacovelli S. Sacchi A. Moretti F. Soddu S. MDM2-regulated degradation of HIPK2 prevents p53Ser46 phosphorylation and DNA damage-induced apoptosis.Mol Cell. 2007; 25: 739-750Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar Recently, HIPK2 has undergone a rapid protein turnover in resting conditions orchestrated by the E3 ubiquitin ligase Siah-1, which, like MDM2, is another p53 target gene. In resting conditions, Siah-1 colocalizes and forms a complex with HIPK2 in nuclear bodies, whereas DNA damage triggers disruption of the complex, via the ATM/ATR pathway, resulting in HIPK2 stabilization and activation.25Winter M. Sombroek D. Dauth I. Moehlenbrink J. Scheuermann K. Crone J. Hofmann T.G. Control of HIPK2 stability by ubiquitin ligase Siah-1 and checkpoint kinases ATM and ATR.Nat Cell Biol. 2008; 10: 812-824Crossref PubMed Scopus (158) Google Scholar Therefore, there is some evidence that HIPK2 is an important actor in DNA damage response, along with p53, which acts as a tumor suppressor gene. Because DNA damage signaling is involved in both colon cancer progression and drug sensitivity, it is tempting to speculate the following: changes in HIPK2 expression could occur during cancer natural history, and HIPK2 expression could affect the drug sensitivity of tumor cells, modulating pharmacologically induced p53 signaling. Thus, it could potentially represent a prognostic marker. To explore HIPK2 expression in colon carcinoma, we selected a collection of 80 pairs of carcinoma and the respective healthy mucosa. By using immunofluorescence and TMA imaging, we developed and validated a novel semiautomatic method (which we coined “TMA cytometry”) for quantifying and analyzing multiple protein expression on archival paraffin-embedded tissue. By applying this technique to our series of colon carcinomas, we simultaneously studied HIPK2 and p53 expression at the cellular level, crossing these data with the mutational status of p53. Moreover, we compared the expression level between the tumor sample and the normal tissue of each patient to explore the possible variations of the protein level occurring during carcinogenesis. Herein, we show that high HIPK2 overexpression is highly and positively correlated with prognosis, irrespective of the p53 mutational status and stage of the disease. Eighty patients with primary colorectal carcinoma, operated on and treated at the Institut Bergonié, Bordeaux, France, between January 1, 1999, and December 31, 2004, were included in the study. At surgery, fragments of tumor and normal mucosa (taken at a distance from the tumor) were frozen quickly in liquid nitrogen and stored at -140°C until extraction. The remaining material was fixed in Holland Bouin's solution and paraffin embedded for pathological evaluation. These frozen and fixed samples were used as paired specimens in the quantification of gene expression by real-time PCR and immunofluorescence analysis, respectively. The clinical characteristics of the series are described in Table 1. At analysis, 39 patients were considered alive without cancer, 8 were alive with cancer, and 4 were alive with another cancer. Twenty-nine patients were dead from cancer (n = 19), complications (n = 3), another cancer (n = 4), or other causes (n = 3).3Iacopetta B. Russo A. Bazan V. Dardanoni G. Gebbia N. Soussi T. Kerr D. Elsaleh H. Soong R. Kandioler D. Janschek E. Kappel S. Lung M. Leung C.S. Ko J.M. Yuen S. Ho J. Leung S.Y. Crapez E. Duffour J. Ychou M. Leahy D.T. O'Donoghue D.P. Agnese V. Cascio S. Di Fede G. Chieco-Bianchi L. Bertorelle R. Belluco C. Giaretti W. Castagnola P. Ricevuto E. Ficorella C. Bosari S. Arizzi C.D. Miyaki M. Onda M. Kampman E. Diergaarde B. Royds J. Lothe R.A. Diep C.B. Meling G.I. Ostrowski J. Trzeciak L. Guzinska-Ustymowicz K. Zalewski B. CapellÁ G.M. Moreno V. Peinado M.A. Lönnroth C. Lundholm K. Sun X.F. Jansson A. Bouzourene H. Hsieh L.L. Tang R. Smith D.R. Allen-Mersh T.G. Khan Z.A. Shorthouse A.J. Silverman M.L. Kato S. Ishioka C. TP53-CRC Collaborative GroupFunctional categories of TP53 mutation in colorectal cancer: results of an International Collaborative Study.Ann Oncol. 2006; 17: 842-847Crossref PubMed Scopus (84) Google Scholar The 5–year overall survival (OS) was 63%, with a median follow–up of 54.6 months. A clear prognostic value of the stage was observed in this series (see Supplemental Figure S1 at http://ajp.amjpathol.org).Table 1Patient CharacteristicsCharacteristicsValueNo. of patients80Age, median (range) (years)66 (31–88)Male/female ratio1:10Primary tumor site Descending colon and sigma-rectum junction55 (68.8) Ascending and transverse colon22 (27.5) Rectum3 (3.8)Tumor stage I and II29 III20 IV29 Unknown2Adjuvant therapy None40 Chemotherapy40Data are given as number (percentage) of patients unless otherwise indicated. Open table in a new tab Data are given as number (percentage) of patients unless otherwise indicated. Tissue cores with a diameter of 0.6 mm were removed from fixed paraffin–embedded tissue blocks and arrayed on a recipient paraffin block using a tissue arrayer (Beecher Instruments Tissue Arrayer, Sun Prairie, WI). Each tumor sample was punched in triplicate, along with a core of nontumoral mucosal tissue punched far away from the tumor. Sections of the array were cut at 5 μm and placed on glass slides. An independent TMA incorporating a series of 30 colorectal cancers with matched normal tissue for eight cases, together with patient survival data, was purchased from US Biomax (CO951, Rockville, MD). IHC with p53 antibody (mouse monoclonal antibody DO–7 Ab, 1:100; Dako, Trappes, France) was performed on a 5–μm fixed paraffin–embedded TMA section mounted on a charged slide. The tissue section was deparaffinized in toluene. After heat-induced proteolytic epitope retrieval in citrate buffer (pH 6.0) using a microwave oven, the primary antibody was incubated for 1 hour at room temperature. Positive reactions were visualized using the avidin–biotin method. Staining was evaluated by visual examination using a semiquantitative approach, assessing the staining intensity with a four–point scale (from zero to three); the percentage of stained cells ranged from 0% to 100%. Double immunofluorescence with p53 (same antibody and conditions as IHC) and HIPK2 (rabbit polyclonal anti–HIPK2 28507 Ab, 1:300; Abcam, Cambridge, UK) was performed on a 5–μm fixed paraffin–embedded TMA section mounted on a charged slide. The tissue section was deparaffinized in toluene. After heat–induced proteolytic epitope retrieval in citrate buffer (pH 6.0) using a microwave oven, primary antibodies were incubated for 1 hour at room temperature. Secondary antibodies [ie, goat anti-rabbit (Alexa Fluor 594 and 532) and goat anti-mouse (Alexa Fluor 488); all from Molecular Probes, Eugene, OR] were diluted in antibody diluent (REAL; Dako) and incubated for 1 hour at room temperature in the dark. The section was washed with PBS three times and incubated with DAPI (1 μg/mL) for 15 minutes in the dark. After dehydration, the section was mounted with Fluoromount–G and stored at 4°C in the dark until analysis. Immunofluorescence signals were collected for each array core with a confocal microscope (LSM 510 META; Zeiss, Göttingen, Germany) using a ×20 objective lens. Images were quantified with the multiwavelength cell scoring application of Metamorph (Molecular Devices, Sunnyvale, CA). A second acquisition and analysis of both TMAs was performed with an automated laser scanning cytometer (iCys; Compucyte, Boston, MA) using phantom segmentation. RNA samples were extracted from fresh–frozen fractions of tumor tissue and normal nontumoral mucosa using a kit (RNeasy Lipid Tissue Qiagen kit, 74804; Qiagen, Valencia, CA), following the manufacturer's instructions. Immediately after elution, RNA samples were cleaned with a DNase inactivation reagent kit (DNase Turbo DNA free; AMBION, Austin, TX), thus avoiding subsequent degradation (data not shown). The RNA samples were treated with DNase (DNase Turbo DNA free), and RNA quality was assessed on a bioanalyzer (Bioanalyseur 2100; Agilent, Santa Clara, CA). Only RNA of high quality [RNA integrity number (RIN) > 7] qualified for the study. First–strand cDNA was synthesized according to the manufacturer's instructions (Reverse Transcriptase Powerscript Clontech, Mountain View, CA). Forty–seven paired samples were available for further analysis. Quantitative analysis was performed on DNA ENGINE OPTICON 2. For standardization, commercial kits for HIPK2 and the housekeeping gene have been used (Applied Biosystems, Carlsbad, CA). Among 10 housekeeping genes tested [ie, GAPDH (glyceraldehyde–3–phosphate dehydrogenase), B2M (β2 microglobulin), PGK1 (phosphorylated glycerate kinase 1), ACTB (actin β), TBP (TATA box binding protein), 18S (18S ribosomal RNA), GUSB (glycuronidase β), HPRT1 (hypoxanthine phosphoribosyl transferase), RPLPO (ribosomal protein large PO), and PPIA (cyclophilin A)], GUSB was the gene that showed less variability in our PCR conditions and was chosen as the housekeeping gene for subsequent quantitative analysis. Each sample was analyzed in triplicate. Paired samples (tumor and normal tissue) were plotted on the same plate. For each sample, fold change (FC) was calculated according to the 2–ΔΔCt method. After evaluation of tumoral cellularity on a frozen H&E–stained section, DNA was extracted from fresh–frozen fractions of tumor tissues using the phenol chloroform method. Of 80 cases, 72 (cellularity >10%) were analyzed. Mutational analysis explored exons 2 to 11 of the p53 gene. Primer sequences (Table 2) were designed with software (Primer 3). PCR was performed in a thermocycler (GeneAmp PCR System 9700; Applied Biosystems). For exons 4 to 11, the PCR mix was composed of 1.5 mmol/L MgCl2, 3.5 μL 10× PCR Gold buffer (Applied Biosystems), dNTP (100 μmol/L each), primer (17.5 pmol each), 1.4 U of AmpliTaq Gold (Applied Biosystems), and 40 ng of DNA (QS, 35 μL). The PCR comprised 5 minutes at 94°C; 45 cycles of 30 seconds at 94°C, 45 seconds at 59°C, and 45 seconds at 72°C; and an elongation phase of 5 minutes at 72°C. For exons 2 to 3, MgCl2 was replaced by 1.5 mmol/L MgSO4, and AmpliTaq Gold was replaced by 1.4 U of Platinium TaqDNA polymerase (Invitrogen) in the PCR mix. The PCR comprised 2 minutes at 94°C; 35 cycles of 30 seconds at 94°C, 45 seconds at 61°C, and 45 seconds at 72°C; and an elongation phase of 5 minutes at 72°C. PCR product sizes were controlled by migration on 2% agarose electrophoresis gel and were purified with columns (GFX PCR DNA kit; Amersham Biosciences, Piscataway, NJ). Bidirectional direct sequencing was performed on the thermocycler (GeneAmp PCR System 9700; Applied Biosystems). Sequencing primers are the same as those of the PCR. Sequencing reaction mix comprises 3.5 μL of 5× sequencing buffer, 0.5 μL of Big Dye Terminator, 12.5 pmol of primer, and 4 μL of PCR products purified and diluted at 1:10 (QS, 20 μL). The sequencing cycle comprised 10 seconds at 94°C and 25 cycles of 5 seconds at 50°C and 4 minutes at 60°C. Purification of sequencing products was performed on a column (DyeEx; Qiagen). Sequence analyses were read on software (SeqScape).Table 2Probe SequencesExonPrimerAmplicon size (bp)Annealing temp (°C)122 and 35′-TCTCAGACACTGGCATGGTG-3′5′-GGGGACAGCATCAAATCATC-3′5006145′-CGTTCTGGTAAGGACAAGGG-3′5′-GGAATCCCAAAGTTCCAAAC-3′484595 and 65′-GTTTGTTTCTTTGCTGCCG-3′5′-TCATGGGGTTATAGGGAGGTC-3′5855975′-CCTCCCCTGCTTGCCAC-3′5′-GAGGTGGATGGGTAGTAGTATGG-3′293598 and 95′-TTGGGAGTAGATGGAGCCTG-3′5′-AAACAGTCAAGAAGAAAACGGC-3′47359105′-TCAAACAATTGTAACTTGAACCATC-3′5′-GGCAGGATGAGAATGGAATC-3′26959115′-GGGAAAAGGGGCACAGAC-3′5′-GCAAGCAAGGGTTCAAAGAC-3′24459 Open table in a new tab To detect inactivating mutations in the TP53 gene, a functional analysis of separated alleles in yeast (FASAY) assay was performed on tumoral RNA extracted for the quantitative RT–PCR analysis, as described by Flaman et al.26Flaman J.M. Frebourg T. Moreau V. Charbonnier F. Martin C. Chappuis P. Sappino A.P. Limacher J.M. Bron L. Benhattar J. Tada M. van Meir E.G. Estreicher A. Iggo R.D. Simple p53 functional assay for screening cell lines, blood, and tumors.Proc Natl Acad Sci U S A. 1995; 92: 3963-3967Crossref PubMed Scopus (429) Google Scholar The transcriptional activity of human p53 in tumor cells is assessed in Saccharomyces cerevisiae, where it activates a p53 target gene (Ade2). This reporter gene is under the control of a promoter that contains the p53 binding site. The mRNA specimens were reverse transcribed with random primers, and part of the TP53 open reading frame comprising exons 4 to 11 was amplified by PCR, leading to a PCR product of 1 kb. The reporter strains were grown and cotransformed with PCR amplicons and a linearized yeast expression vector carrying the 5′ and 3′ ends of the TP53 open reading frame. Activation of the reporter by WT p53 results in white colonies, whereas the mutant p53 produces smaller red colonies. The activity of the p53 mutant was determined by the color of at least 100 colonies per strain. Because of PCR-induced errors or alternatively spliced TP53 mRNA, the FASAY test result was considered negative (functional or WT p53) when <10% of red colonies were detected. The test result was considered doubtful when between 10% and 20% of red colonies were detected. The FASAY test result was considered positive (nonfunctional or mutated p53) for values higher than this cutoff. Positive and negative controls were included in each assay. Survival curves were calculated according to the Kaplan-Meier method; survival analysis was performed using the log-rank test. OS was calculated from the date of diagnosis until death or last follow-up. The Cox proportional hazards model was used for multivariate analysis, and calculation of the hazard ratios and confidence intervals was performed with the ascending step-by-step maximum likelihood method, including only variables significant at P < 0.05 in the univariate analysis. To evaluate the expression of HIPK2 and p53 at the protein level in our tissue collection, we constructed a TMA containing one spot of healthy mucosa and three spots of tumors for each patient. Double immunofluorescence with p53 and HIPK2 antibodies was performed on a 5-μm fixed paraffin-embedded TMA section mounted on a charged slide. The entire TMA section was analyzed by acquisition of p53, HIPK2, and DAPI signals by confocal microscopy (Figure 1, A and B). Each acquired image corresponding to tumoral or normal mucosa tissue core was reviewed for overall tissue content and quality assessment, and the best core for each tumor was chosen for subsequent quantification. Patients were excluded if all three cores were missing or not representative (eg, if they contained only stroma). A manual “virtual microdissection” of the images was performed by excluding the stromal component with a mask to keep only epithelial elements (Figure 1C). Based on DAPI labeling, we submitted each core to a segmentation process that determines the nuclear regions. Each segmented nucleus was quantified for p53 and HIPK2 expression, measuring the mean fluorescence intensity in each channel. Although the analysis routine allows us to automatically score the nuclei as positive or negative using a threshold-based procedure (Figure 1D), we preferred to analyze the distribution of fluorescence intensities of the overall cell populations of spots to avoid generating artifacts linked to the choice of an arbitrary threshold. The fluorescence intensity of each fluorochrome-labeled antibody was measured in individual nuclei, generating a data set for each sample. A mean of 767 cells was quantified in tumor samples (range, 231 to 1324 cells), and a mean of 309 cells was quantified in normal mucosa samples (range, 30 to 682 cells). The TMA from US Biomax contained 30 colorectal cancers (two cores per case), eight of which had matched normal tissue. It was acquired and analyzed with the same procedure as the home-constructed TMA. First, we determined the distribution profiles of p53 nuclear intensities in normal mucosa samples (Figure 2A) and tumoral tissues (Figure 2B). The profiles of normal tissues are highly similar and display a homogeneous gaussian distribution. Tumor samples were more heterogeneous, with a wide range of fluorescence from low to high intensity. Some tumor profiles show the presence of two cell populations, one of them characterized by a high p53 expre
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