The expression vector of LZTFL1 (pcDNA-Flag-LZTFL1) was constructed by subcloning a PCR-amplified insert corresponding to the mouse LZTFL1open-reading-frame (Invitrogen). pTRE2-LZTFL1-ires-EGFP plasmid was constructed by a two-step cloning through PCR and restriction enzyme digestion; the Flag-LZTFL1 fragment from pcDNA-Flag-LZTFL1 was first subcloned into pIres-EGFP vector (Clontech) to yield pLZTFL1-ires-EGFP-ires plasmid. The Flag-LZTFL1-ires-EGFP fragment was then subsequently subcloned into pTRE2 vector (Clontech). GST-LZTFL1 construct (pGEX-kg-LZTFL1) was constructed by subcloning PCR-amplified LZTFL1 fragment into pGEX-kg vector. Construction details are available upon request. The sequences of all cloning products were verified using an automated sequencer.
Generation of LZTFL1 specific antibody
Recombinant Glutathione-S-transferase (GST)-LZTFL1 protein was produced and purified according to a standard protocol (8
). After cleaved and separated from the GST protein, full length LZTFL1 protein was used as the antigen to immunize rabbits (Cocalico Biological, PA).
Cell lines, transfection, and siRNA knock-down
Human intestinal epithelial cell line HT-29, and human breast cancer cell line MCF-7 (ATCC) were maintained in complete medium (DMEM supplemented with 10% fetal bovine serum, 2 mM glutamine, and penicillin-streptomycin). Cells were transfected with combination of plasmids indicated for each experiment using lipofectamine 2000 according to manufacturer’s protocol (Invitrogen). To induce cell differentiation, HT-29 cells were cultured in complete medium supplemented with 3mM sodium butyrate (NaB) for 3 days. For stable transfection, HT-29 cells were transfected with pLZTFL1-ires-EGFP and pIres-EGFP control vector. Cells were selected with G418 for 3–4 weeks.
Three independent LZTFL1 specific siRNA duplexes in TriFecta kit were purchased from IDT (IDT technology) and transfected into C2C12 cells using lipofectamine 2000 (Invitrogen). FITC-labeled non-silencing duplex siRNA was used as the negative control.
Generation of Hela-tet-on cells with doxycycline (Dox)-inducible expression of LZTFL1
Hela-tet-on cells expressing the rtTA (Invitrogen) were maintained in complete medium containing 0.5 mg/ml G418. The cells were transfected with either pTRE2-LZTFL1-ires-EGFP or control pTRE2-ires-EGFP vector using Lipofectamine 2000. GFP was used here as a reporter for the ease of selection. Transfected cells were double selected with hygromycin (0.1 mg/ml) and G418 (0.5 mg/ml). The resistant clones were screened for the induction of GFP expression upon the addition of 1 μg/ml Dox by live fluorescence imaging.
Tissue microarray, immunohistochemistry (IHC), and data analysis
Tissue microarrays were from IMGENEX (San Diego, CA). Slide IMH-326 and IMH-327 contained total of 118 samples of common tumor/cancers. IMH-336 and IMH-337 contained corresponding matched normal/normal adjacent tissues of IMH-326 and IMH-327, respectively. Each individual tumor array contained 8–10 cases. Clinical and pathological information for individual cancer samples was provided by the array manufacturers. IHC staining was carried out following manufacturer’s protocol using LZTFL1 antibody as the primary antibody followed by biotinylated secondary antibody and Streptavidin-horseradish peroxidase. Diaminobenzidine was used as the substrate chromagen and slides were counterstained with hematoxylin. Staining intensity (0: no staining; 1: weak staining; 2: moderate staining; and 3: intense staining) and the proportion of stained cells (0: no staining; 1: <10% staining; 2: between 11% to 33%; 3: between 34% to 66%; and 4: great than 67%) were semi-quantitatively determined following published protocols (9
). All slides were scored by two observers blinded to the pathology and clinical features. Student t-test
was used for statistical analysis. p
was considered statistically significant.
Tumor samples, IHC, patients’ data, and statistical analysis
Paraffin-embedded tumor and corresponding normal tissue sample slides from 84 gastric cancer patients were stained with LZTFL1 antibody using immunohistochemistry and the slides were scored as described above. Normal tissue structures near the cancerous ones in the same histological section or normal corresponding tissue from the same individual in a separate slide were used as positive control. The clinical data were obtained from Department of Surgical Oncology, Sir Run Run Shaw Hospital, China. The patients were enrolled between July 1995 and March 2007. All patients were underwent a curative gastrectomy and none of the patients received preoperative treatments. Total gastrectomy was performed in 10 patients and subtotal gastrectomy in 74 patients. Post-surgery pathological examination showed various tumor types including papillary, mucinous, signet ring, and mucinous adenocarcinoma. All clinical pathological profiles were evaluated in accordance with the criteria of the World Health Organization. Tumor stage was evaluated according to the TNM classification of the 6th edition criteria of the International Union against Cancer.
The patients were followed up until death or until the date of last follow-up on November 30, 2007 and no patient had been lost to follow-up. Thirty-three out of 84 patients (39.2%) died during the follow-up period, and the median follow-up interval was 50.6 months (range from 2.8 to 119.2 months). Informed consents from patients were obtained for the use of paraffin-embedded tumor and normal tissues. This study was approved by the Ethics committee of Sir Run Run Shaw Hospital.
For correlation between LZTFL1 IHC score and clinical parameters, a Pearson r correlation coefficient was calculated using GraphPad Prism Software (GraphPad Software Inc, San Diego, CA). Two-tailed parametric analysis was considered significant when p < 0.05. Survival curves were estimated by the Kaplan-Meir method with log-rank test using GraphPad Prism program.
Soft agar assays and tumorigenicity assays in athymic nude mouse
For soft agar assays, cell aliquots in growth medium mixed with 0.35% soft agar were plated in triplicate onto a 12-well plate with a 0.5% semi-solid agar basal layer. For HT-29 and MCF-7 cells, fresh medium were added every 7 days. For Hela-tet-on cells, fresh medium with and without Dox were added every 3 days. After 4 weeks incubation at 37°C, colonies in the soft agar were photographed and scored under an inverted microscope. For tumorigenicity assays in nude mice, we injected a suspension of 1×107 cells in 0.2 ml PBS subcutaneously into the flank of 6 weeks odd female BALB/C athymic nude mice (NCI). Mice were scored for tumor development and tumor size at each site of injection. Mice were sacrificed and the tumors were weighted when the largest tumor reached 2 cm maximal diameter (at the end of 5 weeks after injection). All animal experiments were performed in accordance with institutional guidelines.
Western blot and immunofluorescence analysis and antibodies
Western blot and immunofluorescence were performed according to standard protocols. Antibodies used in this study are anti-Flag (Sigma), anti-cadherin (Cell Signaling), anti-β-catenin and anti-GAPDH (Santa Cruz), and anti-LZTFL1 (this study). Confocal immunofluorescence images were taken on Zeiss LSM510.
In vitro migration assay
Parental, EGFP-, and LZTFL1- expressing Heta-tet-on cells were cultured in the presence and absence of Dox for 48 hrs, washed, trypsinized, and suspended in DMEM plus 0.1% BSA. Approximately 1×105 cells in 100 μl DMEM/0.1%BSA were placed into the upper chamber of a 24-well Transwell cell culture plate (Corning Costar). The lower chamber contained DMEM with 5% FBS as a chemoattractant. After 8 hrs of incubation, migrated cells on the lower surface were stained with hematoxylin and counted under a microscope.