Ovarian tissues and cell lines
Ovarian cancer and normal control tissues were obtained from The University of Texas M. D. Anderson Cancer Center tumor tissue bank with the approval of the Institutional Review Board. The ovarian cancer cell lines NIH:OVCAR3, SKOV3, OV-90, TOV-112D, TOV-21G, and PA-1 were purchased from the American Type Culture Collection (Manassas, VA). NIH:OVCAR3 cells were maintained in RPMI 1640 medium supplemented with 20% fetal bovine serum (FBS); SKOV3 and PA-1 cell were maintained in McCoy's 5a medium and Dulbecco's modified Eagle medium (DMEM)/F12, respectively, supplemented with 10% FBS; OV-90, TOV-112D, and TOV-21G cells were maintained in a 1:1 mixture of MCDB 105 medium and medium 199, supplemented with 15% FBS. All cells were kept at 37°C in a humidified atmosphere with 5% CO2. Media were routinely changed every 3 days.
Construction of progression tissue microarray for ovarian serous tumors
Formalin-fixed, paraffin-embedded archival tissue blocks from ovarian cancer patients who had undergone surgery at The University of Texas M. D. Anderson Cancer Center between 1990 and 2001 were used to construct progression tissue microarrays according to previously described methods . The progression tissue microarray consisted of normal ovarian surface epithelium from 6 individuals, serous borderline tumors from 17 patients, and invasive serous carcinomas from 40 patients. Tissue cores with a diameter of 1.0 mm were obtained from each sample and assembled into two separate tissue array blocks.
Polyclonal antibody against IGFBP2 (c-18; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) was used in the immunohistochemistry studies. This antibody is specific and does not cross-react with other isoforms of IGFBP. A standard indirect immunoperoxidase procedure (ABC-Elite; Vector Laboratories, Burlingame, CA) was used for all stains. In brief, antigen retrieval was performed by first placing unstained slides in a steamer for 25 min. The antibody against IGFBP2 (in a 1:1000 dilution) was overlaid on the tissue sections of tissue arrays, and incubation was performed at 4°C overnight. Secondary antibody incubation was performed at room temperature for 60 min. Mayer's hematoxylin nuclear stain was used as a counterstain. Staining intensity was graded on a 0–3 scale, where 0 = no staining as assessed by staining with anti-goat secondary antibody alone, 1 = weak (<10%), 2 = moderate (10–50%), and 3 = strong (50–100%). The results of the immunohistochemistry studies were statistically analyzed using a Mann-Whitney nonparametric U test.
Stable clone establishment
To establish stable cell lines that overexpressed IGFBP2, we transfected SKOV3 ovarian cancer cell lines with a pcDNA3.1 expression vector encoding IGFBP2 cDNA using FuGENE6 reagent (Roche Diagnostics Corporation, Indianapolis, IN). Transfected cells were subsequently selected in the presence of G418 (300 μg/ml) for 5 weeks. The expression of IGFBP2 clones was determined from western blots of cell extracts with anti-IGFBP2 antibody (C-18). Two vector-transfected cell lines and three IGFBP2 stable cell lines were used in this study. Established stable cells were maintained without antibiotics.
Western blot analysis
Equal amounts of proteins from the total cell lysates was separated by 10% SDS-PAGE and transferred electrophoretically to a Hybond ECL nitrocellulose membrane (Amersham Pharmacia Biotech, Chicago, IL). The membrane was blocked in 5% skim milk in 1× PBS and probed with a 1:1000 dilution of a goat polyclonal anti human IGFBP2 (C-18) overnight at 4°C. An enhanced chemiluminescence kit (ECL; Amersham Pharmacia Biotech, Piscataway, NJ) was used to visualize the proteins.
In vitro chemoinvasion assay
We used 24-well BioCoat Matrigel invasion chambers (Becton Dickinson Labware, Bedford, MA) with an 8-μm pore polycarbonate filter coated with Matrigel to measure chemoinvasion. The lower compartment contained 0.75 ml of medium with 0.5% FBS as a chemoattractant. In the upper compartment, 5 × 104 to 2 × 105 cells/well were placed in triplicate wells and incubated for 22 h at 37°C in a humidified incubator with 5% CO2. After incubation, the cells that had passed through the filter into the lower wells were stained with Giemsa (Fisher Scientific, Orangeburg, NY) and counted by analyzing images under a microscope using software program as described previously . All assays were repeated at least three times. Student's t-test was used to analyze the differences in the invasion rates between control cell lines and stable cell lines. A p value of <0.05 was considered statistically significant.
Four different siRNA molecules designed for IGFBP2 mRNA and siRNA molecules for Lamin A/C and negative control were synthesized and purified (Qiagen, Valencia, CA). siRNA that targets Lamin A/C and siRNA that bears no homology with relevant human genes was used as a negative control.
AATGGCGATGACCACTCAGAA was the target sequence for siRNA1; AAGGGTGGCAAGCATCACCTT was the target sequence for siRNA2; AAGCGCCGGGACGCCGAGTAT was the target sequence for siRNA3; AACCTCAAACAGTGCAAGATG was the target sequence for siRNA4; AACTGGACTTCCAGAAGAACA was the target sequence for Lamin A/C; and AATTCTCCGAACGTGTCACGT was the target sequence for the negative control. siRNAs were dissolved in siRNA suspension buffer to a final concentration of 20 μM, and the mixture was heated to 90°C for 1 min and incubated at 37°C for 60 min. PA-1 ovarian cancer cells (2 × 105) were plated to a 6-well plate and allowed to adhere for 24 h; the confluency of the cell monolayer at the time of transfection was 40–60%. 5 μg of siRNA and 15 μl of RNAiFect Transfection Reagent (Qiagen, Valencia, CA) was used. The cells were incubated under normal cell culture conditions. All assays were performed 72 h after treatment.