Animal crosses and tumor material
The animal material was derived from crosses between BDII/Han females and males from two non-susceptible rat strains, BN/Han and SPRDCu3/Han, where at first an F1 progeny was produced. Subsequently, an F2 offspring by brother/sister mating of the F1 progeny, and a backcross progeny (N1), by crossing the F1 males to BDII females, were produced. The female progeny was palpated twice each week for identification of uterine tumors. Animals suspected to have tumors were euthanized and the tumor tissue surgically removed, subjected to pathological characterization and subsequently used to establishment of cell cultures [29]. In this study we have investigated cell lines established from tissues pathologically classified as endometrial adenocarcinomas (EAC) and from tissues of normal/pre-malignant endometrium (NME). RUT cell lines originate from tumors developed in the F1 and F2 progeny and NUT cell lines originate from the tumors in the backcross progeny (Table 1).
In vitro cell culture conditions
Primary cell cultures established from the EAC tumors were propagated in Dulbecco's modified Eagle medium, supplemented with 100 IU/100 μg/ml penicillin/streptomycin, L-glutamine, MEM amino acids, MEM Non Essential Amino acids, MEM Vitamins solution and 10% heat-inactivated fetal bovine serum, for 3-5 passages in order to obtain the required amount of cells. The NME cell lines were cultured under the same conditions, but in medium containing 20% fetal bovine serum. The cells were grown at 37°C in an atmosphere of 95% humidity and 5% CO2 and harvested by trypsinizination at a confluence of 80-90% (approx 1x106 cells).
RNA extraction
Total RNA was extracted from the harvested cells of the different endometrial rat cell lines with a KingFisher mL Instrument (Thermo Electron Corporation, USA) according to the manufacturer's protocol (MagAttract Tissue Mini M48 Kit, Qiagen). RNA was spectrophotometrically quantified (NanoDrop technologies, USA).
Quantitative PCR (qPCR) of Gpx3 and Met in cell lines from adenocarcinomas (EAC) and normal/premalignant tissue (NME)
A total of 14 EAC and 2 NME cell lines were used in qPCR analysis with GAPDH as an endogenous control and Universal Rat Reference RNA, Agilent Technologies, Inc as a calibrator (Table 1). RT-PCR was performed using High Capacity cDNA Reverse Transcription Kit according to the manufacturer's protocol (Applied Bio-systems). Template cDNA was added to TaqMan Universal Master Mix (AB; Applied Biosystems, Foster City, CA, USA) in a 12.5 μl reaction with specific pre-designed probes for the Gpx3 and Met (Applied Biosystems). Reactions were performed in duplicates and threshold cycle number was averaged. Relative gene expression quantification was calculated according to the comparative Ct method using GAPDH as an endogenous control and Universal Rat reference RNA (Stratagene) as calibrator. The relative quantitative gene expression, were determined as follows: 2-(Ct sample-Ct calibrator), where Ct values of the calibrator and sample are determined by subtracting the Ct value of the target gene from the value of the GAPDH gene.
Bisulfite treatment and methylation-specific PCR
One μg DNA from the susceptible rat strain BDII, 15 EAC and 2 NME cell lines were denatured, sodium bisulfite treated and purified using Epitect Bisulfite Kit according to the manufacturer's protocol (Qiagen) (Table 1). As a positive control, DNA from an endometrial cell line (NUT43) with a normal expression of Gpx3, and thus unmethylated was treated with methylase and subsequently treated with sodium bisulfite. The modified tumor/control DNA was used as template for methylation-specific PCR. Methylation specific primers (MSP) were designed using the publicly available MethPrimer program http://www.urogene.org/methprimer/[30]. The bisulphite modification of DNA converts unmethylated cytosines to uracils, whereas methylated cytosines will remain unchanged. Bisulphite treated DNA was amplified with either methylation specific or un-methylation specific primer sets. PCR was carried out in a final 25 μl volume containing 50 ng of template DNA The mixture was heated at 94°C for 1 min and then subjected to 35 cycles of 94°C, 55°C and 72°C and a final extension at 72°C for 7 min. The PCR product was analyzed on a 2% agarose gel with appropriate size marker and the absence or presence of PCR product were detected.
5-aza-2´-deoxycytidine and trichostatin A (TSA) treatment in rat endometrial tumor cell lines
Two EAC cell lines (NUT12 and NUT81) with confirmed biallelic methylated promoter status of Gpx3 were treated with 5-aza-2´-deoxycytidine and trichostatin A (Sigma). Cells were grown in a medium containing 2.5 μM 5-aza-cytidine for 96 hours, with the medium and drug being replaced every 24 hours and the addition of 300 nM TSA was added for the last 16 hours. After 96 hours, the drugs were removed and total RNA for Gpx3 RT-PCR expression analysis was extracted using AllPrep RNA/DNA Mini Kit according to the manufacturer's protocol (Qiagen).
Hydrogen peroxide measurements
The intra- and extra-cellular amount of hydrogen peroxide in the endometrial cell lines were measured using the Amplex® Red Peroxide/Peroxidase Hydrogen assay kit according to the manufacturer's protocol (Molecular Probes, Invitrogen). The three cell lines investigated (NUT12, NUT43, NUT56) were seeded to a 96 wells plate (Corning) with an initial number of 5000 cells/well (Table 1). In each well, the amount of hydrogen peroxide was measured post 72 hours of incubation, as described above. Each cell line was replicated 20 times. The cells were lysated by adding RIPA buffer (25 mM TRIS-HCl pH 7.6, 150 mM NaCl, 1% deoxycholate, 0, 1% SDS and 1% NP40) followed by incubation on ice for 30 minutes. Intra- and extra-cellular H2O2 concentrations were assessed by pooling 50 μl of cell lysate with 50 μl of used cell culture media.
Development of FISH probes for Gpx3
DNA from the pre-malignant cell line, NUT43, with a normal expression of Gpx3 was used to generate a probe that represented only the Gpx3 gene. Six primer pairs, specific for the Gpx3 gene, with a product size of approximately 600 base pair each (Table 5), were designed by using the Primer 3 program available on the internet: http://fokker.wi.mit.edu/primer3/input.htm. Amplification was performed by PCR and carried out in a final 25 μl volume containing 100 ng of template DNA. The mixture was heated at 94°C for 1 min and then subjected to 35 cycles of 94°C, 58°C and 72°C and a final extension at 72°C for 7 min. Sizes of the PCR products were determined on a 2% agarose gel with an appropriate size marker. The PCR products were then purified by Mini Elute PCR purification kit (QIAGEN) and the concentration of DNA was measured by NanoDrop (NanoDrop Technologies, USA) amplified product. The amplified sequences were then pooled and fluorescently labeled by dNTP in DOP-PCR and subsequently used as a Gpx3 specific probe in FISH.
One probe, which was used as positive control, was developed from a RNO2 BAC clone (CH230-397A17 from BACPAC Resources Center, Oakland, California). The BAC DNA was amplified by DOP-PCR as follows. The reactions were performed in a final volume of 25 μl and with a BAC DNA concentration of 20 ng/μl. The mixture was heated at 94°C for 1 min and then subjected to 35 cycles of 94°C, 55°C and 72°C and a final extension at 72°C for 7 min. The product was verified on a 2% agarose gel with an appropriate size marker. For FISH, the Nick Translation kit from Abbott molecular was used according to the manufactures protocol.
Human material
A total of 30 EACs in FIGO grade I-III (10 tumors from each grade embedded in archival formalin fixed paraffin (FFPE) were used in the study. Apart from the endometrial tumors, 21 benign endometrial tissues were collected, and reference material from lung was used in the normalization process (Table 2). All samples were anonymous. A pathologist marked the tumor area in samples in the hematoxylin and eosin slide. Using a Tissue Micro Array-equipment (Pathology Devices), 3-4 cores (∅0.6 mm) of tumor tissue was punched out from the paraffin block. After standard tissue sample deparaffinization using xylene and alcohols, samples were lyzed in a Tris-chloride, EDTA, sodium dodecyl sulfate (SDS) and proteinase K containing buffer. RNA was then extracted and used for the real time qPCR.
Quantitative PCR (qPCR) of GPX3 and MET in FIGO grade I-III human EACs
Total RNA was extracted and used for qPCR according to the same procedure as for the rat samples.
Statistical analysis
For statistical evaluations of Ct values for differences among replicates we applied paired samples t-test and for comparisons of normal and malignant tissues independent sample t-test was applied. (PASW Statistics 18, SPSS Inc, Chicago, USA). In both tests the null hypotheses were assuming no differences between replicates, and no differences between tissue types respectively. The Pearson correlation test was performed to check for correlation between the expression of Gpx3 and Met. The significance levels were set to P < 0.5 in all statistical tests.
