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PTOV-1, a Novel Protein Overexpressed in Prostate Cancer, Shuttles between the Cytoplasm and the Nucleus and Promotes Entry into the S Phase of the Cell Division Cycle

2003; Elsevier BV; Volume: 162; Issue: 3 Linguagem: Inglês

10.1016/s0002-9440(10)63885-0

1525-2191

Anna Santamaría, Pedro L. Fernández, Xavier Farré, Patricia Benedit, Jaume Reventós, Juan Moróte, Rosanna Paciucci, Timothy M. Thomson,

Prostate Cancer Treatment and Research

PTOV1 was recently identified as a novel gene and protein during a differential display screening for genes overexpressed in prostate cancer. The PTOV1 protein consists of two novel protein domains arranged in tandem, without significant similarities to known protein motifs. By immunohistochemical analysis, we have found that PTOV1 is overexpressed in 71% of 38 prostate carcinomas and in 80% of samples with prostate intraepithelial neoplasia. High levels of PTOV1 in tumors correlated significantly with proliferative index, as assessed by Ki67 immunoreactivity, and associated with a nuclear localization of the protein, suggesting a functional relationship between PTOV1 overexpression, proliferative status, and nuclear localization. In quiescent cultured prostate tumor cells, PTOV1 localized to the cytoplasm, being excluded from nuclei. After serum stimulation, PTOV1 partially translocated to the nucleus at the beginning of the S phase. At the end of mitosis, PTOV1 exited the nucleus. Transient transfection of chimeric green fluorescent protein-PTOV1 forced the entry of cells into the S phase of the cell cycle, as shown by double fluorescent imaging for green fluorescent protein and for Ki67, and also by flow cytometry. This was accompanied by greatly increased levels of cyclin D1 protein in the transfected cells. These observations suggest that overexpression of PTOV1 can contribute to the proliferative status of prostate tumor cells and thus to their biological behavior. PTOV1 was recently identified as a novel gene and protein during a differential display screening for genes overexpressed in prostate cancer. The PTOV1 protein consists of two novel protein domains arranged in tandem, without significant similarities to known protein motifs. By immunohistochemical analysis, we have found that PTOV1 is overexpressed in 71% of 38 prostate carcinomas and in 80% of samples with prostate intraepithelial neoplasia. High levels of PTOV1 in tumors correlated significantly with proliferative index, as assessed by Ki67 immunoreactivity, and associated with a nuclear localization of the protein, suggesting a functional relationship between PTOV1 overexpression, proliferative status, and nuclear localization. In quiescent cultured prostate tumor cells, PTOV1 localized to the cytoplasm, being excluded from nuclei. After serum stimulation, PTOV1 partially translocated to the nucleus at the beginning of the S phase. At the end of mitosis, PTOV1 exited the nucleus. Transient transfection of chimeric green fluorescent protein-PTOV1 forced the entry of cells into the S phase of the cell cycle, as shown by double fluorescent imaging for green fluorescent protein and for Ki67, and also by flow cytometry. This was accompanied by greatly increased levels of cyclin D1 protein in the transfected cells. These observations suggest that overexpression of PTOV1 can contribute to the proliferative status of prostate tumor cells and thus to their biological behavior. Prostate adenocarcinoma is the second most prevalent cancer among males in Western countries, with an incidence that increases in direct proportion with age. A large body of evidence indicates that activation of the pathways regulated by androgen receptor plays a central role in the development and malignant progression of prostate cancer.1Jenster G The role of the androgen receptor in the development and progression of prostate cancer.Semin Oncol. 1999; 26: 407-421PubMed Google Scholar Because this is a physiologically regulated pathway, the question remains as to the underlying mechanisms that can direct the activation of the androgen receptor-signaling routes toward undue responses, eventually leading to malignant transformation of the prostate epithelial cells. In a few instances of primary tumors, mutations in the androgen receptor have been found that permit its activation by steroids other than androgens, or even other stimuli, independent of steroids.2Grossmann ME Huang H Tindall DJ Androgen receptor signaling in androgen-refractory prostate cancer.J Natl Cancer Inst. 2001; 93: 1687-1697Crossref PubMed Scopus (466) Google Scholar Most frequently, however, androgenreceptor mutations are a late event in the progression of prostate cancer,3Marcelli M Ittmann M Mariani S Sutherland R Nigam R Murthy L Zhao Y DiConcini D Puxeddu E Esen A Eastham J Weigel NL Lamb DJ Androgen receptor mutations in prostate cancer.Cancer Res. 2000; 60: 944-949PubMed Google Scholar and appear to be a major mechanism, along with androgen receptor gene amplification and overexpression,4Linja MJ Savinainen KJ Saramaki OR Tammela TL Vessella RL Visakorpi T Amplification and overexpression of androgen receptor gene in hormone-refractory prostate cancer.Cancer Res. 2001; 61: 3550-3555PubMed Google Scholar by which these tumors become insensitive to androgens, and independent from these hormones for their growth.5Taplin ME Bubley GJ Shuster TD Frantz ME Spooner AE Ogata GK Keer HN Balk SP Mutation of the androgen-receptor gene in metastatic androgen-independent prostate cancer.N Engl J Med. 1995; 332: 1393-1398Crossref PubMed Scopus (1021) Google Scholar This hormone-independent state is usually accompanied by an increase in the capacity of tumor cells to invade surrounding tissues and to form distant metastases.6Klein KA Reiter RE Redula J Moradi H Zhu XL Brothman AR Lamb DJ Marcelli M Belldegrun A Witte ON Sawyers CL Progression of metastatic human prostate cancer to androgen independence in immunodeficient SCID mice.Nat Med. 1997; 3: 402-408Crossref PubMed Scopus (334) Google Scholar In consequence, in the majority of prostate cancers other mechanisms must be invoked to explain the early events leading to the induction of the neoplastic phenotype. Over the past few years, genetic and biochemical approaches have identified a number of molecular alterations that are associated with different stages of prostate tumorigenesis.7Blok LJ Kumar MV Tindall DJ Isolation of cDNAs that are differentially expressed between androgen-dependent and androgen-independent prostate carcinoma cells using differential display PCR.Prostate. 1995; 26: 213-224Crossref PubMed Scopus (49) Google Scholar, 8Chang GT Blok LJ Steenbeek M Veldscholte J van Weerden WM van Steenbrugge GJ Brinkmann AO Differentially expressed genes in androgen-dependent and -independent prostate carcinomas.Cancer Res. 1997; 57: 3814-3822Google Scholar, 9Lucas S De Smet C Arden KC Viars CS Lethe B Lurquin C Boon T Identification of a new MAGE gene with tumor-specific expression by representational difference analysis.Cancer Res. 1998; 58: 743-752PubMed Google Scholar, 10Stubbs AP Abel PD Golding M Bhangal G Wang Q Waxman J Stamp GW Lalani EN Differentially expressed genes in hormone refractory prostate cancer: association with chromosomal regions involved with genetic aberrations.Am J Pathol. 1999; 154: 1335-1343Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar Systematic approaches usingmicroarray-based global transcriptome analysis have also attempted to identify markers and pathways altered in prostate cancer.11Lin B Ferguson C White JT Wang S Vessella R True LD Hood L Nelson PS Prostate-localized and androgen-regulated expression of the membrane-bound serine protease TMPRSS2.Cancer Res. 1999; 59: 4180-4184PubMed Google Scholar, 12Xu J Stolk JA Zhang X Silva SJ Houghton RL Matsumura M Vedvick TS Leslie KB Badaro R Reed SG Identification of differentially expressed genes in human prostate cancer using subtraction and microarray.Cancer Res. 2000; 60: 1677-1682PubMed Google Scholar, 13Luo JH Yu YP Cleply K Lin F Deflavia P Dhir R Finkelstein S Michalopoulos G Becich M Gene expression analysis of prostate cancers.Mol Carcinog. 2001; 33: 25-35Crossref Scopus (209) Google Scholar, 14Chaib H Rubin MA Mucci NR Li L Taylor JMG Day ML Rhim JS MaCoska JA Activated in prostate cancer: a PDZ domain-containing protein highly expressed in human primary prostate tumors.Cancer Res. 2001; 61: 2390-2394PubMed Google Scholar, 15Chetcuti A Margan S Mann S Russell P Handeslman D Rogers J Dong Q Identification of differentially expressed genes in organ-confined prostate cancer by gene expression array.Prostate. 2001; 47: 132-140Crossref PubMed Scopus (63) Google Scholar, 16Dhanasekaran SM Barrette TR Ghosh D Shah R Varambally S Kurachi K Pienta KJ Rubin MA Chinnaiyan AM Delineation of prognostic biomarkers in prostate cancer.Nature. 2001; 412: 822-826Crossref PubMed Scopus (1415) Google Scholar, 17Welsh JB Sapinoso LM Su AI Kern SG Wang-Rodriguez J Moskaluk CA Frierson Jr, HF Hampton GM Analysis of gene expression identifies candidate markers and pharmacological targets in prostate cancer.Cancer Res. 2001; 61: 5974-5978PubMed Google Scholar, 18Singh D Febbo PG Ross K Jackson DG Manola J Ladd C Tamayo P Renshaw AA D'Amico AV Richie JP Lander ES Loda M Kantoff PW Golub TR Sellers WR Gene expression correlates of clinical prostate cancer behavior.Cancer Cell. 2002; 1: 203-209Abstract Full Text Full Text PDF PubMed Scopus (1981) Google Scholar, 19Ernst T Hargenhahn M Kenzelmann M Cohen CD Bomrouhi M Weninger A Klären R Gröne EF Wiesel M Güdemann C Küstler J Schott W Staehler G Kretzler M Hollstein M Gröne H-J Decrease and gain of gene expression are equally discriminatory markers for prostate carcinoma.Am J Pathol. 2002; 160: 2169-2180Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar These studies have identified novel markers of early-stage prostate neoplasia,14Chaib H Rubin MA Mucci NR Li L Taylor JMG Day ML Rhim JS MaCoska JA Activated in prostate cancer: a PDZ domain-containing protein highly expressed in human primary prostate tumors.Cancer Res. 2001; 61: 2390-2394PubMed Google Scholar as well as transcriptional signatures of advanced disease and poor prognosis.18Singh D Febbo PG Ross K Jackson DG Manola J Ladd C Tamayo P Renshaw AA D'Amico AV Richie JP Lander ES Loda M Kantoff PW Golub TR Sellers WR Gene expression correlates of clinical prostate cancer behavior.Cancer Cell. 2002; 1: 203-209Abstract Full Text Full Text PDF PubMed Scopus (1981) Google Scholar In general, the possible biological significance of the altered expression of the genes identified in such analyses has not been addressed experimentally. In our laboratory, we have recently identified a new protein, which we have designated PTOV1, that is overexpressed in a significant proportion of prostate adenocarcinomas.20Benedit P Paciucci R Thomson TM Valeri M Nadal M Càceres C de Torres I Estivill X Lozano JJ Morote J Reventos J PTOV1, a novel protein overexpressed in prostate cancer containing a new class of protein homology blocks.Oncogene. 2001; 20: 1455-1464Crossref PubMed Scopus (57) Google Scholar Recent global transcriptional profile analyses by others17Welsh JB Sapinoso LM Su AI Kern SG Wang-Rodriguez J Moskaluk CA Frierson Jr, HF Hampton GM Analysis of gene expression identifies candidate markers and pharmacological targets in prostate cancer.Cancer Res. 2001; 61: 5974-5978PubMed Google Scholar have found that PTOV1 is one of the genes most discriminant between the normal and carcinomatous prostate. Importantly, we have found that PTOV1 is also overexpressed in early neoplastic lesions, the prostate intraepithelial neoplasias.20Benedit P Paciucci R Thomson TM Valeri M Nadal M Càceres C de Torres I Estivill X Lozano JJ Morote J Reventos J PTOV1, a novel protein overexpressed in prostate cancer containing a new class of protein homology blocks.Oncogene. 2001; 20: 1455-1464Crossref PubMed Scopus (57) Google Scholar At present, the function of this protein is not known, nor can it be deduced or suggested from its sequence, because it does not contain any known protein motifs.20Benedit P Paciucci R Thomson TM Valeri M Nadal M Càceres C de Torres I Estivill X Lozano JJ Morote J Reventos J PTOV1, a novel protein overexpressed in prostate cancer containing a new class of protein homology blocks.Oncogene. 2001; 20: 1455-1464Crossref PubMed Scopus (57) Google Scholar In this report we present an extended immunohistochemical study on samples from 38 prostate adenocarcinomas showing a correlation between proliferating tumor cells and levels of PTOV1 expression in vivo. We also describe cell-cycle-associated changes in subcellular localization of PTOV1 in prostate cancer cell lines, and the consequences on the cell cycle of its overexpression by transient transfection. Our observations suggest that overexpression of PTOV1 can have a direct influence on the proliferative state of prostate cells. Rabbit antibodies to human PTOV120Benedit P Paciucci R Thomson TM Valeri M Nadal M Càceres C de Torres I Estivill X Lozano JJ Morote J Reventos J PTOV1, a novel protein overexpressed in prostate cancer containing a new class of protein homology blocks.Oncogene. 2001; 20: 1455-1464Crossref PubMed Scopus (57) Google Scholar were affinity purified using Sulfolink columns from Pierce (Rockford, IL), on which the immunizing peptide was covalently immobilized. Monoclonal anti-Ki67 antibody was from Immunotek (Marseille, France), anti-p27Kip1 antibody was from Novocastra (Edinburgh, UK) and anti-cyclin D1 was from Santa Cruz Biotechnology (Santa Cruz, CA). Peroxidase-coupled anti-rabbit antibodies, tetramethyl-rhodamine isothiocyanate-coupled anti-rabbit antibodies were from Sigma (Alcobendas, Madrid, Spain) and fluorescein isothiocyanate-coupled anti-mouse antibodies were from DAKO (Glostrup, Denmark). A total of 38 prostate adenocarcinomas from radical prostatectomies for T2 to T3 tumors were obtained from the files of the Department of Pathology of the Hospital Clinic of Barcelona. The mean patient age in this study was 63.5 ± 5.81 years (median, 64 years; range, 45 to 75 years). The mean preoperative serum prostate-specific antigen (PSA) was 11.50 ± 7.88 ng/ml (median, 9.6 ng/ml; range, 1.29 to 38 ng/ml). The Gleason sum score assigned to the radical prostatectomy specimens ranged from 4 to 9 and was evaluated as grade ≤6 (low, 17 patients) versus ≥7 (high, 21 patients). Normal or hyperplastic prostatic tissue could be evaluated in 29 of the cases as well as 10 high-grade prostatic intraepithelial neoplasia (PIN) lesions. Also, normal prostatic tissue from six patients who underwent cystoprostatectomy for bladder tumors were included in the study. Two-μm sections from multitissue blocks21Fernández PL Nayach I Fernández E Fresno L Palacín A Farré X Campo E Cardesa A Tissue macroarrays ("microchops") for gene expression analysis.Virchows Arch. 2001; 438: 591-594Crossref PubMed Scopus (15) Google Scholar harboring 6-mm-diameter selected fragments were deparaffinized in xylene, and rehydrated with graded alcohols, water, and phosphate-buffered saline (PBS). Antigen unmasking was performed with pressure cooker heating in ethylenediaminetetraacetic acid buffer, pH 7, for 2 minutes, slides were allowed to cool down for 5 minutes and were sequentially incubated in vertical humid chambers with PBS (3 minutes) background suppressor (DAKO S3022, 30 minutes at room-temperature) and the primary antibody (2 μg/ml overnight, humid chamber at 4°C). Slides were then rinsed twice in PBS and endogenous peroxidase-quenched with 2% hydrogen peroxide in methyl alcohol for 30 minutes. After rinsing twice in PBS, slides were incubated for 30 minutes with Envision anti-rabbit antibody (Envision, DAKO). The staining was developed with substrate chromogen solution (Envision, DAKO) and diaminobenzidine for 3 to 5 minutes. The slides were counterstained with Gill-I hematoxylin for 1 minute, dehydrated, and mounted with DPX solution. Negative controls consisted in the use of 100 μl of the primary antibody previously incubated at 37°C for 1 hour with 0.16 mg of the purified peptide, the latter previously resuspended in 0.1 N of acetic acid, and used as above in parallel with similar slides with nonadsorbed primary antibody. The use of such preadsorbed primary antibody gave clean negative results in all cases tested (not shown). A semiquantitative gradation of the intensity was performed with intensities ranging from weak (intensity level 1) to very strong (intensity level 4). A case was considered positive when >10% of the cells showed unequivocal staining (levels ≥1). Both cytoplasmic and nuclear staining were recorded. A similar procedure was followed for detection of Ki67 except that incubation of the primary antibody was for 30 minutes at room temperature and was preceded by antigen unmasking in citrate buffer. Ki67 was evaluated by counting the total of positive cells in all tumor fragments of the same size. A correction factor was used when the fragments were only partially involved by tumor. Staining for p27Kip1 was performed with the streptavidin-alkaline phosphatase method (Biogenex, San Ramon, CA) as previously described22Fernández PL Arce Y Farré X Martínez A Nadal A Rey MJ Peiró N Campo E Cardesa A Expression of p27/kip1 is down-regulated in human prostate carcinoma progression.J Pathol. 1999; 187: 563-566Crossref PubMed Scopus (47) Google Scholar and the percentage of positive cells was recorded. All cell lines were obtained from the American Type Culture Collection (Rockville, MD). Cell lines PC-3, LNCaP, and COS7 were maintained in RPMI 1640 medium (Life Technologies, Inc., Grand Island, NY) supplemented with 10% heat-inactivated fetal bovine serum (Life Technologies, Inc.) at 37°C in an atmosphere of 5% CO2. The prostate cell line PZ-HPV-7 was maintained in Keratinocyte-Serum-Free Medium (Life Technologies, Inc.) supplemented with 5 ng/ml of human recombinant epidermal growth factor and 50 μg/ml of bovine pituitary extract. Immunocytochemistry for confocal microscopy was performed as described.20Benedit P Paciucci R Thomson TM Valeri M Nadal M Càceres C de Torres I Estivill X Lozano JJ Morote J Reventos J PTOV1, a novel protein overexpressed in prostate cancer containing a new class of protein homology blocks.Oncogene. 2001; 20: 1455-1464Crossref PubMed Scopus (57) Google Scholar Briefly, cells cultured on coverslips were washed with cold PBS, fixed with 4% paraformadehyde for 10 minutes, incubated with 50 mmol/L of NH4Cl for 30 minutes, and permeabilized with PBS/1% bovine serum albumin/0.1% saponin for 30 minutes. Samples were sequentially incubated with primary antibody diluted in permeabilization buffer for 2 hours, and secondary antibody for 1 hour, and mounted with Immuno-Fluore Mounting Medium (ICN, Costa Mesa, CA). Fluorescence was visualized by inverted fluorescence microscope DM IRBE (Leica, Wetzlar, Germany) and captured by a TCS-NT argon/krypton confocal laser (Leica). Cultured cells, washed with cold PBS, were lysed with Laemmli sample buffer [50 mmol/L Tris-HCl, pH 6.8, 2% (w/v) sodium dodecyl sulfate, 10% (v/v) glycerol, β-mercaptoethanol] heated at 95°C and centrifuged at 10,000 × g. Fifty to 100 μg of cell lysate were electrophoresed and transferred to nitrocellulose. Membranes were blocked for 1 hour with blocking buffer (5% nonfat dry milk in PBS/1% Tween-20) and incubated for 2 hours with anti-PTOV1 (5 μg/ml in blocking buffer), washed, and incubated with horseradish peroxidase-conjugated goat anti-rabbit Ig. Reactivity was detected with a chemiluminescent substrate (Amersham, Buckinghamshire, UK). For sequential Western blotting, membranes were stripped at 50°C according to the manufacturer's instructions. Constructs for the expression of chimeric green fluorescent protein (GFP)-PTOV1 with GFP at the amino or carboxy termini of PTOV1 have been described previously.20Benedit P Paciucci R Thomson TM Valeri M Nadal M Càceres C de Torres I Estivill X Lozano JJ Morote J Reventos J PTOV1, a novel protein overexpressed in prostate cancer containing a new class of protein homology blocks.Oncogene. 2001; 20: 1455-1464Crossref PubMed Scopus (57) Google Scholar Transient transfections were done with Lipofectamine (Life Technologies, Inc.). Twenty-four or 48 hours after transfection, cells were washed, fixed in 4% paraformaldehyde, processed for immunofluorescence with anti-PTOV1, mounted, and analyzed by confocal microscopy. Control transfections were performed with the vector pEGFP alone, for the expression of nonchimeric GFP, which consistently yielded a diffuse fluorescence distributed throughout the cells, without any subcellular specific localization. Cells were fixed in 70% ethanol in PBS at −20°C for at least 1 hour, washed several times with cold PBS, and treated with RNaseA (50 μg/ml) for 30 minutes at 37°C and incubated with propidium iodide (30 μg/ml). Cells were subsequently analyzed on a Coulter Epics XL flow cytometer (Coulter, Miami, FL) for GFP fluorescence (detection filter set at 525 nm) and DNA content (filter set at 675 nm). Cell aggregates were gated out, and 10,000 events were analyzed. For each cell type analyzed, untransfected cells were used to establish a threshold for green fluorescence (up to 102 arbitrary fluorescence units in a typical case), and which was taken as a threshold for positivity. GFP-positive and -negative populations were analyzed separately for DNA content and assigned to specific cell-cycle phases by applying the Multicycle cell-cycle analysis software (Phoenix Flow Systems, San Diego, CA). In our initial studies on tumor samples, most prostate adenocarcinomas analyzed by immunohistochemistry expressed high levels of PTOV1, without a correlation between these levels and clinicopathological parameters such as histological grade or clinical stage.20Benedit P Paciucci R Thomson TM Valeri M Nadal M Càceres C de Torres I Estivill X Lozano JJ Morote J Reventos J PTOV1, a novel protein overexpressed in prostate cancer containing a new class of protein homology blocks.Oncogene. 2001; 20: 1455-1464Crossref PubMed Scopus (57) Google Scholar To test whether other parameters of in vivo biological activity, more directly associated with proliferative status, correlate with levels of PTOV1, we analyzed samples from 38 prostate adenocarcinomas and 6 normal prostates for the expression of PTOV1 and two other markers, the antigen Ki67, as an indicator of proliferating cells,23Gerdes J Lemke H Baisch H Wacker H-H Schwab U Stein H Cell cycle analysis of a cell proliferation-associated human nuclear antigen defined by the monoclonal antibody Ki-67.J Immunol. 1984; 133: 1710-1715PubMed Google Scholar and the cyclin-dependent kinase inhibitor p27Kip1, a regulator of the G1 to S transition24Sherr C Roberts JM CDK inhibitors: positive and negative regulators of G1-phase progression.Genes Dev. 1999; 13: 1501-1512Crossref PubMed Scopus (5072) Google Scholar that is frequently down-regulated in prostate cancer.22Fernández PL Arce Y Farré X Martínez A Nadal A Rey MJ Peiró N Campo E Cardesa A Expression of p27/kip1 is down-regulated in human prostate carcinoma progression.J Pathol. 1999; 187: 563-566Crossref PubMed Scopus (47) Google Scholar, 25Guo Y Lklar GN Borkowski A Kyprianou N Loss of the cyclin-dependent kinase inhibitor p27(Kip1) protein in human prostate cancer correlates with tumor grade.Clin Cancer Res. 1997; 3: 2269-2274PubMed Google Scholar, 26Cordon-Cardo C Koff A Drobnjak M Capidieci P Osman I Millard SS Gaudin PB Fazzari M Zhang ZF Massague J Scher HI Distinct altered patterns of p27KIP1 gene expression in benign prostatic hyperplasia and prostatic carcinoma.J Natl Cancer Inst. 1998; 90: 1284-1291Crossref PubMed Scopus (242) Google Scholar Immunohistochemistry for these markers was performed in serial or near-serial sections. As previously reported, PTOV1 was mostly undetectable or showed a weak (level 1) cytoplasmic staining in normal prostate glandular epithelial cells (Figure 1). Interestingly, a more intense (level 3) staining was seen in sporadic luminal cells in isolated glands, which was mostly cytoplasmic but in some cases also nuclear. Staining of serial sections showed that these PTOV1-positive cells most likely corresponded to chromogranin A-positive cells, a marker for neuroendocrine cells (data not shown). Also, a very strong PTOV1 expression (levels 3 to 4) was observed in endothelial cells, erythrocytes (Figure 1A), and ganglion cells of vegetative ganglia. Therefore, most normal glandular epithelial cells usually do not express PTOV1, or do so at low levels, although several types of nonepithelial cells present in normal prostate structures can express PTOV1 at high levels. Of the 38 adenocarcinoma samples analyzed, 27 (71%) overexpressed (ie, expression levels 2 to 3) PTOV1 in cells of carcinomatous appearance (Figure 1 and Table 1). The majority of the specimens positive for PTOV1 staining showed a homogeneous distribution of positive tumor cells throughout the carcinomatous areas, with >80% positive cells. Overexpression of PTOV1 was limited to the cytoplasm of carcinomatous cells in 16 cases, whereas it was strong (level 3) both in the nucleus and the cytoplasm in the remaining 11 cases (Table 2). Two cases were an exception to this situation in that tumor cells had a strong nuclear staining in combination with a weak cytoplasmic staining for PTOV1. There was no obvious association between gland morphology (differentiation) or topographical area of the tumor (periphery versus central) with the levels or the pattern of PTOV1 expression. Ten tumors also contained abnormal glands with morphological characteristics of PIN. Of these, eight showed strong cytoplasmic or cytoplasmic and nuclear staining for PTOV1 (Figure 1 and Table 1, Table 2) without significant differences in intensity between basal or luminal cells, and also contained a higher number of Ki67-positive cells. In the tumor samples analyzed, the expression levels of PTOV1 in the epithelial cells of glands with a normal morphology (BPZ) were either very low (level 1) or undetectable (equal to or below background staining) (Figure 1 and Table 1).Table 1Summary of Immunohistochemical Analyses of PTOV1 Expression in Prostate Cancer and Normal ProstatePTOV1Level 0*Level 0, no detectable staining; level 1, low; and level 2/3, high levels of expression (see Materials and Methods). (%)Level 1 (%)Level 2/3 (%)P†P values obtained by comparisons with BPZ.Tumor Carcinoma011/38 (29)27/38 (71)0.0001 PIN02/10 (20)8/10 (80)0.0001 BPZ6/29 (20.7)22/29 (75.8)1/29 (3.4)Normal3/6 (50)3/6‡Focal expression in isolated glands (<10% of cells). (50)0* Level 0, no detectable staining; level 1, low; and level 2/3, high levels of expression (see Materials and Methods).† P values obtained by comparisons with BPZ.‡ Focal expression in isolated glands (<10% of cells). Open table in a new tab Table 2Expression of Ki67 and p27 in Relation to PTOV1 Levels and Subcellular Localization in Prostate TumorsPTOV1 level 1PTOV1 level 2/3PKi67 index*Total PTOV1, average of Ki67 index for all samples; PTOV1 C only, average of Ki67 index for samples with PTOV1 localized to the cytoplasm only; PTOV1 C and N, average of Ki67 index for samples with PTOV1 localized both to the cytoplasm and the nucleus.Total PTOV142.574.50.013PTOV1 C only42770.015PTOV1 C and N083.60.010†P value obtained by comparison with Ki67 values of tumors expressing low levels of PTOV1, with PTOV1 localized to the cytoplasm only.p27 (% positive cells)26270.714* Total PTOV1, average of Ki67 index for all samples; PTOV1 C only, average of Ki67 index for samples with PTOV1 localized to the cytoplasm only; PTOV1 C and N, average of Ki67 index for samples with PTOV1 localized both to the cytoplasm and the nucleus.† P value obtained by comparison with Ki67 values of tumors expressing low levels of PTOV1, with PTOV1 localized to the cytoplasm only. Open table in a new tab The same samples were analyzed by immunohistochemistry for the expression of Ki67 and p27Kip1. Expression of Ki67 was assessed in the form of an index, calculated as a function of positive nuclei per total nuclei in sections of comparable size (see Materials and Methods). Normal prostate epithelium did not express Ki67 at significant levels (index <1). In tumor samples, areas of normal morphology did not show Ki67 staining or did so in very few cells (index <1), whereas the carcinomatous areas from the same samples had indexes that ranged from 4 to more than 100 (Figure 1F). When these indexes were compared with levels of expression of PTOV1, a significant correlation (P = 0.013) was found between Ki67 index and levels of PTOV1 (Table 2), indicative of an association of high levels of PTOV1 with an active proliferative status. Of interest, all of the tumor samples with nuclear localization of PTOV1 showed high levels (levels 2 to 3) of expression of PTOV1 and had elevated Ki67 indexes (Table 2). Expression of p27Kip1 protein was evaluated in 18 of the 38 tumors. As shown in Table 2, no significant correlation was observed between levels of expression of PTOV1 and levels of p27Kip1. Patients with overexpression of PTOV1 (levels 2 or 3) showed higher PSA serum levels (12.62 ng/ml) compared to patients with low levels (level 1) (8.57 ng/ml), although this association did not reach statistically significant values (P = 0.056). When subcellular localization of PTOV1 was considered, independently of its levels of expression, patients whose samples showed nuclear expression of PTOV1 had significantly (P = 0.019) higher preoperative serum PSA levels (13.83 ng/ml) than did patients whose samples had an exclusively cytoplasmic localization of PTOV1 (8.74 ng/ml). Increased levels of expression of PTOV1 were not associated with a higher Gleason score (≥7, not shown), confirming our previous observations.20Benedit P Paciucci R Thomson TM Valeri M Nadal M Càceres C de Torres I Estivill X Lozano JJ Morote J Reventos J PTOV1, a novel protein overexpressed in prostate cancer containing a new class of protein homology blocks.Oncogene. 2001; 20: 1455-1464Crossref PubMed Scopus (57) Google Scholar In conclusion