Affiliations of authors: S. C. Mok, G. K. Yiu, Department of Obstetrics, Gynecology, and Reproductive Biology, Brigham and Women's Hospital, Harvard Medical School, Boston, MA; J. Chao, Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston; S. Skates, Gillette Center for Women's Cancer, Dana-Farber Cancer Institute, and Biostatistics Center, Massachusetts General Hospital, Boston; K. Wong, Department of Pediatrics, Texas Children's Hospital, Baylor College of Medicine, Houston; M. G. Muto, R. S. Berkowitz, D. W. Cramer, Department of Obstetrics, Gynecology, and Reproductive Biology, Brigham and Women's Hospital, Harvard Medical School, and Gillette Center for Women's Cancer, Dana-Farber Cancer Institute.
Correspondence to: Samuel C. Mok, Ph.D., Laboratory of Gynecologic Oncology, Brigham and Women's Hospital, 221 Longwood Ave., BLI 449, Boston, MA 02115 (e-mail: scmok{at}rics.bwh.harvard.edu).
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ABSTRACT |
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INTRODUCTION |
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Microarray technology permits the simultaneous comparison of the expression of thousands of genes in samples to allow identification of those that are differentially expressed. The technique has been applied to the molecular classification of tumors (35) and may also be able to identify overexpressed complementary DNA (cDNA) corresponding to secretory proteins that might serve as serum markers for cancer. In this article, we describe the application of microarray technology to identify novel molecular markers for ovarian cancer and explore the potential clinical value of one of the candidate markers (called prostasin) thus identified.
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MATERIALS AND METHODS |
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All patient-derived biologic specimens were collected and archived under protocols approved by the Human Subjects Committee of the Brigham and Women's Hospital, Boston, MA, or were approved for study under guidelines covering discarded human materials. Ovarian tissue and cells were freshly collected from women undergoing surgery at the Brigham and Women's Hospital for a diagnosis of primary ovarian cancer or from control subjects having hysterectomy and oophorectomy for benign disease. Cultures of normal human ovarian surface epithelial (HOSE) cells were established by scraping the surface of the ovary and growing recovered cells in a mixture of medium 199 and MCDB105 medium supplemented with 10% fetal calf serum (Sigma Chemical Co., St. Louis, MO) as described previously (6). The following seven normal HOSE cells were used: HOSE17, HOSE636, HOSE642, HOSE697, HOSE713, HOSE726, and HOSE730. Ovarian cancer cell lines were established by recovery from ascites fluid or explanted from solid tumors as described previously (6). The following 10 ovarian cancer cell lines were used: OVCA3, OVCA420, OVCA429, OVCA432, OVCA433, OVCA633, CAOV3, DOV13, ALST, and SKOV3. All of the cell cultures and cell lines were established in the Laboratory of Gynecologic Oncology, Brigham and Woman's Hospital, with the exception of SKOV3, which was purchased from the American Type Culture Collection, Manassas, VA. RNA was extracted from individual or pooled cell lines by using a micro RNA extraction kit as described by the manufacturer (Qiagen, Valencia, CA) and quantified by fluorometry (Gemini Bio-Products, Inc., Calabasas, VA).
Serum specimens from women with ovarian cancer, other gynecologic cancers, and benign gynecologic disorders requiring hysterectomy and from nondiseased normal women were obtained from discarded specimens and from discarded specimens that were archived during the period from 1983 through 1988 or from specimens collected under more recent protocols since 1996. The archived samples were collected from several studies, from 1983 through 1988, assessing the performance of CA 125 in a variety of diagnostic circumstances, including gynecologically normal subjects as well as subjects having exploratory surgery for pelvic masses that proved to be ovarian, cervical, or endometrial cancer or a benign disease such as fibroid tumors (79). These archived specimens were stored at -70 °C. However, during relocation of the Laboratory of Gynecologic Oncology, thawing was known to have occurred once for some of the archived specimens. Archived specimens that had been obtained preoperatively from the case patients and from the surgical control subjects were identified and recovered. The recently collected specimens are those being obtained with written informed consent as part of ongoing studies of ovarian cancer sponsored by the Obstetrics/Gynecology Epidemiology Unit and the Laboratory of Gynecologic Oncology, Brigham and Women's Hospital. These specimens were obtained within the past 5 years and were stored at -70 °C without any incident of thawing. In both specimen banks, serum from case patients with ovarian cancer and serum from control patients were collected concurrently.
Microarray Probe and Hybridization
The MICROMAXTM human cDNA microarray system I (NEN Life Science Products, Inc., Boston, MA), which contains 2400 known human cDNA on a slide 1 inch x 3 inches, was used in this study. Biotin-labeled cDNA was generated from 3 µg of total RNA that was pooled from HOSE17, HOSE636, and HOSE642 cells. Dinitrophenyl-labeled cDNA was generated from 3 µg of total RNA that was pooled from ovarian cancer cell lines OVCA420, OVCA433, and SKOV3. Before the cDNA reaction, equal amounts (5 ng) of Arabidopsis control RNA were added to each batch of the RNA samples for the normalization of hybridization signals. The biotin-labeled cDNA and the dinitrophenyl-labeled cDNA were mixed, dried, and resuspended in 20 µL of hybridization buffer (5x standard saline citrate [SSC], 0.1% sodium dodecyl sulfate [SDS], and salmon sperm DNA at 0.1 mg/mL [1x SSC = 0.15 M NaCl0.15 M sodium citrate, pH 7]). This mixture was added to the cDNA microarray and was covered with a coverslip. Hybridization was carried out overnight at 65 °C inside a hybridization cassette (Telechem, Inc., Sunnyvale, CA).
After hybridization, the microarray was washed with 30 mL of 0.5x SSC0.01% SDS, with 30 mL of 0.06x SSC0.01% SDS, and then with 30 mL of 0.06x SSC alone. The hybridization signal from biotin-labeled cDNA was amplified with streptavidinhorseradish peroxidase and a fluorescent dye, Cy5TM-tyramide (NEN Life Science Products, Inc.), and the hybridization signal from the dinitrophenyl-labeled cDNA was amplified with anti-dinitrophenylhorseradish peroxidase and another fluorescent dye, Cy3TM-tyramide (NEN Life Science Products, Inc.). After signal amplification and a posthybridization wash in TNT buffer (i.e., 0.1 M TrisHCl [pH 7.5]0.15 M NaCl0.15% Tween 20), the microarray was air-dried, and signal amplification was detected with a laser scanner.
Laser detection of the Cy3 signal (derived from ovarian cancer cells) and the Cy5 signal (derived from HOSE cells) on the microarray was acquired with a confocal laser reader, ScanArray3000 (GSI Lumonics, Watertown, MA). Separate scans were taken for each fluor at a pixel size of 10 µm. cDNA derived from the added Arabidopsis RNA hybridized to 12 specific spots on the microarray, which were composed of DNA sequences obtained from four different Arabidopsis, expressed sequence tags in triplicate (NEN Life Science Products, Inc.). Cy3 and Cy5 signals from these 12 spots should theoretically be equal and were used to normalize the different efficiencies in labeling and detection with the two fluors. The fluorescence signal intensity and the ratio of the signals from Cy3 and Cy5 for each of the 2400 cDNAs were analyzed by the software Imagene 3.0 (Biodiscovery Inc., Los Angeles, CA).
Real-Time Quantitative Reverse TranscriptionPolymerase Chain Reaction
Real-time reverse transcriptionpolymerase chain reaction (RTPCR) was performed in duplicate by using primer sets specific for the overexpressed gene encoding the secretory protein called prostasin (forward primer = 5'-ACTTGAGCCACTCCTTCCTTCAG-3'; reverse primer = 5'-CTGATGGTCCCAAAAAGCACAC-3') and a housekeeping gene, GADPH, in an ABI PRISM 5700 Sequence Detector (PE Applied Biosystems, Foster City, CA). RNA was first extracted from normal ovarian epithelial cell cultures (HOSE697, HOSE713, HOSE726, and HOSE730) and from 10 ovarian carcinoma cell lines (OVCA3, OVCA420, OVCA429, OVCA432, OVCA433, OVCA633, CAOV3, DOV13, SKOV3, and ALST). cDNA was generated from 1 µg of total RNA by using the TaqMan RT reagents containing 1x TaqMan reverse transcriptase buffer, 5.5 mM MgCl2, all four deoxyribonucleoside triphosphates (each at 500 µM), 2.5 µM random hexamer, MultiScribe reverse transcriptase (PE Applied Biosystems) at 1.25 U/µL, and RNasin (Promega Corp., Madison, WI) at 0.4 U/µL in 100 µL. The reaction was incubated at 25 °C for 10 minutes, at 48 °C for 30 minutes, and finally at 95 °C for 5 minutes. A total of 1 µg of cDNA was used in a 20-µL PCR mixture containing 1x SYBR® PCR buffer, 3 mM MgCl2, all four deoxyribonucleoside triphosphates (each at 0.8 mM), and AmpliTaq Gold (all from PE Applied Biosystems). The cDNAs were then amplified by denaturation for 10 minutes at 95 °C, followed by 40 PCR cycles of denaturation at 95 °C for 15 seconds and annealingextension at 60 °C for 1 minute. The changes in fluorescence of the SYBR Green I dye in every cycle were monitored by ABI5700 system software, and the threshold cycle (CT), which represents the PCR cycle at which an increase in reporter fluorescence above a baseline signal can first be detected for each reaction, was calculated as described by Heid et al. (10). The relative amount of PCR products generated from each primer set was determined on the basis of the CT value. GAPDH was used to normalize the quantity of RNA used. Its CT value was then subtracted from that of each target gene to obtain a CT value. The difference (
CT) between the
CT values of the samples for each gene target and the
CT value of a calibrator (sample 697), which served as a physiologic reference, was determined. The relative quantitative value was expressed as 2-
CT. For confirmation of the specificity of the PCR, PCR products were subjected to electrophoresis on a 1.2% agarose gel. A single PCR product with the expected size should be observed in samples that express the gene of interest.
Immunohistochemical Localization of Prostasin
Immunostaining with an anti-prostasin antibody was performed on sections prepared from two normal ovaries, from two serous borderline ovarian tumors, and from two grade 1, two grade 2, and two grade 3 serous ovarian cystadenocarcinomas. This rabbit polyclonal antibody (provided by Dr. Julie Chao's laboratory) also used in the serum assay was prepared from prostasin purified from human seminal fluid as described previously (11). Tissues were fixed in 4% paraformaldehyde and embedded in paraffin. Sections (5 µm) were cut, mounted on Superfrost/Plus microscopic slides (Fisher Scientific, Pittsburgh, PA), and incubated at 50 °C overnight. They were then transferred to Tris-buffered saline (TBS) and quenched in 0.2% H2O2 for 20 minutes. After quenching, the sections were washed in TBS for 20 minutes, incubated with normal horse serum for 20 minutes, and then incubated with anti-prostasin polyclonal antibody (diluted 1 : 400) at room temperature for 1 hour. The slides were then washed in TBS for 10 minutes, incubated with diluted biotinylated secondary horse anti-rabbit antibody solution for 30 minutes, washed again in TBS for 10 minutes, incubated with avidinbiotin complex reagent (Vector Laboratories, Inc., Burlingame, CA) for 30 minutes, and washed in TBS for 10 minutes. Stain development was performed for 5 minutes by use of the diaminobenzidine kit (Vector Laboratories, Inc.). Finally, the sections were washed in water for 10 minutes. They were then counterstained with hematoxylin, dehydrated with an ascending series of alcohol solutions, cleared in xylene, and mounted in Permount (Fisher Scientific). The specificity of the staining was confirmed by using preimmunization rabbit serum and by preabsorbing the antibody with the purified peptide (60 mg/mL; Genosys, Woodlands, TX) or prostasin for 2 hours at 37 °C before applying the adsorbed antiserum to the sections.
Measurement of Prostasin and CA 125 in Sera
Sera were available from a total of 201 subjects (64 case patients with ovarian cancer and 137 control subjects, including 24 with other gynecologic cancers, 42 with benign gynecologic diseases, and 71 with no known gynecologic diseases). In all of the case patients and in the 68 control subjects who had surgery, preoperative specimens were available. Serum levels of immunoreactive human prostasin were determined by enzyme-linked immunosorbent assay (ELISA) prepared with the previously described antibody to human prostasin (11). Microtiter plates (96-well) were coated with anti-prostasin immunoglobulin G (IgG) (1 µg/mL, 100 µL per well) overnight at 4 °C. Purified prostasin standards (0.1610 ng) or samples were added to individual wells in a total volume of 100 µL of phosphate-buffered saline containing 0.05% Tween 20 and 0.5% gelatin (dilution buffer) and incubated at 37 °C for 90 minutes. Biotin-labeled anti-human prostasin IgG was added in each well at a concentration of 1 µg/mL in a total of 100 µL and incubated at 37 °C for 60 minutes. Peroxidaseavidin at a concentration of 1 µL/mL in a total volume of 100 µL was added and incubated at 37 °C for 30 minutes. The color reaction was performed by adding to each well 100 µL of freshly prepared substrate solution [0.03% 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) and 0.03% H2O2 in 0.1 M sodium citrate (pH 4.3)] and incubating the mixture at room temperature for 30 minutes. The plates were read at 405 nm with a plate reader (Titertek Instruments Inc., Huntsville, AL).
For 37 case patients with nonmucinous ovarian cancers and for 100 control subjects (about 70% of all subjects), a CA 125 level had been determined previously (from the same specimens) and was available in our database for a comparison standard. These measurements had been performed with the original CA 125 radioimmunoassay from Centocor (Malvern, PA), and the assays were not repeated for this study.
Statistical Analysis
Univariate comparisons for quantitative variables between normal and cancer cell lines or between case and control sera were made by using Student's t test. For the analysis of serum levels, adjustment for potential confounding variables, such as the subject's age, year of collection, and whether the specimen had undergone freezing and thawing, was carried out by using general linear modeling. Logistic regression analysis was used to determine the statistical significance of both prostasin and CA 125 as a predictor of case status. Paired Student's t test was used to compare the change in postoperative prostasin levels from preoperative levels. Pearson correlation coefficients were calculated between CA 125 and prostasin. Because the distributions of prostasin and CA 125 were skewed positively, log-transformed values were used in the statistical tests. All of the analyses were performed with the Statistical Analyses System (SAS Institute, Cary, NC) or Splus (Insightful Corporation, Seattle, WA). Analyses with a P value of .05 or less were considered to be statistically significant. All statistical tests were two-sided, and all confidence intervals (CIs) are 95%.
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RESULTS |
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DISCUSSION |
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Chemically, prostasin is a trypsin-like serine proteinase with an apparent molecular mass of 40 kd (11). The 20-amino acid sequence at the amino terminus of prostasin is 50%55% identical to that of human -tryptase, elastase 2A and 2B, chymotrypsin, acrosin, and the catalytic chains of hepsin, plasma kallikrein, and coagulation factor XI (11). Like the enzymatic activity of other serine proteinases, the enzymatic activity of prostasin is dependent on a catalytic triad of the amino acids histidine, aspartic acid, and serine (15), which are present in motifs that are highly conserved among serine proteinases (16). Similar to acrosin and testisin (17,18), prostasin is likely to be a membrane-anchored protein because there is a putative transmembrane domain of 19 amino acids at the carboxyl terminus that is believed to anchor the protein to the plasma membrane of prostate epithelial cells, from which it is released by cleavage. In view of prostasin's homology with other serine proteases, it is not surprising that several serine proteases are elevated in patients with ovarian cancer; these proteases include certain kallikreins such as protease M/kallikrein 6 (19), prostate-specific antigen (20), hepsin (21), and testisin (18). Our current understanding of prostasin does not provide an explanation of why it or other serine proteinases might be overexpressed in ovarian cancer.
Although we believe that we have demonstrated prostasin's potential value as a biomarker for ovarian cancer, this study has several potential limitations. First, our sample size is relatively small and not ideal for demonstrating the value of prostasin as a screening marker. Although all blood samples were drawn preoperatively, all of the women with ovarian cancer had symptomatic disease and about 55% had stage III disease or greater. In addition, a majority of the sera were obtained from an archived bank, and some of the specimens had undergone freezing and thawing. We observed no tendency for freezing and thawing to produce lower prostasin levels; however, there was evidence that specimens kept in the freezer longer may have had lower values for prostasin. For this reason, we adjusted for length of freezer storage in the multivariate model, and this adjustment did not negate the difference between case patients with ovarian cancer and control subjects. Our sample did not address prostasin's potential as a marker for tumor recurrence because sera preceding recurrences were not available. Finally, we can only partially address how prostasin might be complementary to other markers for ovarian cancer. It may be complementary to CA 125 as suggested by the low correlation between the two.
We believe that our study also demonstrates the potential value of microarray technology to identify tumor biomarkers that may have clinical usefulness. In this study, we used the MICROMAX cDNA microarray system that contained the 2400 genes with known function at the time of the development of this chip (13). We selected this chip because it was the only chip available at the time that we began this research. Subsequently, microarrays with an even larger collection of genes or expression-sequencing tags have become available, such as the GeneChipTM U95 Set (Affymetrix Inc., Santa Clara, CA) and the GeneAlbumTM GEMTM 16 (IncyteGenomics, San Francisco, CA), which represent more than 50 000 genes or expression-sequencing tags. Besides choice of the microarray chip, an important technical issue is the source of the tumor and the normal cDNA for comparison. In this study, we pooled cDNA from several cancer cell lines and compared it with cDNA from normal HOSE cells. The principal advantage of using cell lines is that they provide an abundant source of RNA from the precise cell types involved in epithelial ovarian cancer. Their chief disadvantage is that they are a step removed from the actual cancer in vivo and would not detect genes that might be differentially expressed in the stroma of ovarian cancer specimens and that might be important.
Our study also provides a case illustration of the types of validation studies, which are necessary once a differentially expressed gene has been identified through microarray technology. Overexpression of a gene should be confirmed in individual cell lines or in tissues from individuals with cancer or from normal control subjects. Under ideal circumstances, an assay will be available to detect the gene product through either immunostaining of tissues or detection in sera. At each step, these validation studies must be consistent with differential expression associated with the cancer. Thus, although our preliminary study described 30 genes overexpressed in ovarian cancer cell lines when we used the MICROMAX cDNA microarray system (13), we have focused on one of the candidate genes in this study, which we believe has satisfied these additional elements of validation.
In conclusion, we believe that our study demonstrates the potential value of the powerful new technology of cDNA microarray in identifying overexpressed genes in ovarian cancer, and we suggest that prostasin may be a biomarker with clinical potential. Thus, larger studies that can yield more precise estimates of the sensitivity and specificity of prostasin, either alone or in combination with CA 125, will be necessary.
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NOTES |
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Manuscript received December 26, 2000; revised July 23, 2001; accepted August 1, 2001.
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