Informed consent was obtained from patients prior to surgical resection of adrenal tissue according to a protocol approved by the local Institutional Review Board and Yale Pathology Tissue services. Tissue was flash-frozen in liquid nitrogen and stored at -80°C until processed for study. Specimens displaying unequivocal histopathological characteristics of ACC (n
7), ACA (n
8), and normal adrenal cortical tissue (n
6) samples were selected for use in the study. Consecutive unstained and Hematoxylin & Eosin (H&E) stained 5 μm sections of tumor and normal formalin-fixed paraffin embedded (FFPE) tissue samples were obtained from Yale Tissue Pathology services. All samples were evaluated by experienced endocrine pathologists before processing.
DNA, RNA, and Protein preparation
Genomic DNA from tissue samples were isolated using the DNeasy blood and tissue kit from Qiagen (Valencia, CA). Total RNA from the samples were isolated using the RNeasy Mini Kit (Qiagen, Valencia, CA,) after rotor-stator homogenization, per the manufacturer’s recommendations. Quantity and quality of prepared DNA and RNA was assessed by spectrophotometry (NanoDrop Technologies, Inc., Thermo Fischer, Waltham, MA) and 1% agarose gel electrophoresis. Total protein from cultured cells were extracted using Laemmli buffer (BioRad, Hercules, CA) and protein concentrations were measured using the Pierce BCA Protein assay Kit (Thermo Scientific, Rockford, IL) and Multimax detection system (Promega, Madison, WI), per the manufacturer’s instructions.
Gene expression analysis
Total RNA (100 ng) was reverse transcribed using Superscript III reverse transcriptase (Applied Biosystems, Rockville, MD). Quantitative real-time PCR (qPCR) was performed on triplicate samples using TaqMan PCR master mix with the FAM flurophore and probe/primer pairs specific to RASSF1A (Applied Biosystems, Rockville, MD) according to the manufacturer’s cycling conditions using CFX96 thermal cyclers (Bio-Rad, Hercules, CA). Gene expression levels were normalized to the averages of expression levels of beta-actin and TATA-binding protein probe/primer pairs (Applied Biosystems, Rockville, MD). The Cycle Threshold (CT) values were calculated using the recommended Livak method (Bio-Rad, Hercules, CA).
Methylation status of CpG Island A of the RASSF1A promoter was assessed by MethylScreen technology using the Epitect methyl II PCR assay (Qiagen, Valencia, CA). Briefly, 125 ng of genomic DNA was mock-digested or digested with methylation-sensitive and methylation-dependent restriction enzymes individually or together, and the methylation status of the target sequence was measured using real-time qPCR with probes specific to the target promoter sequences. The amplification results that corresponds to >60% digestion by methylation-dependent restriction enzyme represents Hypermethylated sequences and 0% digestion indicates completely unmethylated DNA. Any amount of digestion between 0% and 60% represents the ‘intermediate methylation’ fraction. The Cycle Threshold (CT) values were converted into percentages of unmethylated, intermediately-methylated and hypermethylated CpG values, using a quantitation algorithm provided by the manufacturer (EpiTect Methyl II PCR Assay Handbook – Qiagen, Valencia, CA).
Immunohistochemical (IHC) and Immunofluorescence (IF) detection
Five μm-thick FFPE sections were processed for immunohistochemistry according to the protocol recommended by the manufacturer of 3,3’Diaminobenzidine (DAB) substrate (BD Biosciences, San Jose, CA). Mouse anti-RASSF1A (1:100) primary antibody (Abcam, Cambridge, MA), goat anti-mouse/Biotin antibody (Santa Cruz Biotech., Santa Cruz, CA) and streptavidin-HRP (Life technologies, Rockville, MD) were used prior to DAB substrate development and detection (BD Biosciences, San Jose, CA). Nikon Eclipse E600 microscope with Spot 3.5 program was used to take photomicrographs at a total magnification of 400X. Immunofluorescence detection of RASSF1A proteins and microtubules were carried out as described
]. Mouse anti-RASSF1A mAb (1:100; Abcam, Cambridge, MA) or goat anti-RASSF1A goat polyclonal (1:200; Santa CruZ Biotech, Santa Cruz, CA) primary antibodies and anti-goat FITC, anti-goat TR, anti-mouse FITC, or anti-mouse TR secondary antibodies (1:1000; all from Santa Cruz Biotech., Santa Cruz, CA) were used followed by ultracruz mounting agent containing 4’,6-diamidino-2-phenylindole - DAPI (Santa Cruz Biotech., Santa Cruz, CA) for immunodetection. Anti-DDK (DYKDDDDK epitope) monoclonal antibody (1:200; Origene, Rockville, MD) was used for detecting DDK-tagged RASSF1A
in transfected cells. Rhodamine Phalloidin, a high affinity F-actin probe conjugated to the red-orange fluorescent dye, tetramethylrhodamine (TRITC) (Biotium, Hayward, CA) was used for immunofluorescence detection of microtubules. Dilutions and incubations were carried out per the manufacturer’s recommendations. Zeiss AX10 confocal microscope with AxioVision 4.8 program was used for immunofluorescence analysis and photomicrographs were taken at a total magnification of 1000X.
Cell culture, expression vectors, transfections, and Western blot detection
The human ACC cell line SW-13 was purchased from American Type Cell Collection (Manassas, VA) and was maintained under sterile conditions in DMEM supplemented with 10% certified fetal bovine serum and 10,000 U/mL penicillin/streptomycin (all from Life Technologies, Inc., Rockville, MD) in a standard humidified incubator at 37.0 C and 5% CO2. Myc-DDK tagged pCMV6-Entry, pCMV6-Entry/RASSF1A, and pCMC6-Entry/RASSF1A/A133S plasmid vectors (Origene, Rockville, MD) were used for transfection. Transfected SW-13 cells were designated SW-13/V representing pCMV vector alone, SW-13/A representing pCMV-RASSF1A, and SW-13/AM representing pCMV-RASSF1A/A133S mutant.
Transient transfection was carried out using Lipofectamine2000 according to the manufacturer’s recommendations (Life Technologies, Inc., Rockville, MD) in 6-well plates with a starting density of 80,000 cells/well. Transfected cells were allowed to grow for 6 days, to test the effect of RASSF1A and RASSF1A/A133S mutant gene expression on growth potential and survival of SW-13 cells. Total cell numbers and viability were calculated by staining cells with 0.4% Trypan Blue (GIBCO-BRL, Life Technologies, Inc., Rockville, MD) and manual counting using a counting chamber (Housser Scientific Co., PA). Experiments were performed in triplicate, and parallel plates with cells growing on glass coverslips were used to determine transfection efficiency and continued expression of transfected genes by indirect immunofluorescence.
Stable clones expressing RASSF1A and RASSF1A/A133S were selected in 800 μg/ml G-418 (Life technologies Inc., Rockville, MD) containing growth medium. Multiple clones were then pooled into populations to avoid expression variability between clones. Established populations (designated SW-13/V, SW-13/A, and SW-13/AM representing pCMV vector alone, pCMV-RASSF1A, and pCMV-RASSF1A/A133S mutant, respectively) were used to determine the effects of constitutive expression of RASSF1A or RASSF1A/A133S on SW-13 cell’s malignant behavior. Expression of transfected genes were confirmed via Western blotting using anti-DDK mAb for (1:1000; Origene, MD), anti-RASSF1A mAb (1:500, Abcam, MA), anti-mouse-HRP (Santa Cruz Biotech., CA), mini-PROTEAN TGX gel, PVDF blotting membrane (BioRad, Hercules, CA), and enhanced chemiluminescnce (ECL) detection reagents (Pierce Thermo Scientific, Rockford, IL) according to the manufacturer’s protocols. Equal protein loading between lanes were confirmed by staining PVDF membranes after chemiluminescence detection.
Cell migration, invasion, and clonogenicity assays
Stable SW-13/V, SW-13/A or SW-13/AM cells were allowed to invade through a Matrigel layer from upper chambers containing serum-free medium to the lower chamber containing 10% FBS medium in BDBiocoat matrigel invasion chambers (BD Biosciences, Bedford, MA). After 24 hours, the Matrigel was removed, and invaded cells were fixed in 3.7% formaldehyde/PBS for 10 minutes, stained with 0.5% crystal violet for 2 hours, and counted using 10X magnification with a light microscope. The Matrigel invasion assay was performed twice in duplicate chambers. In the migration assay, the stably-transfected cells were allowed to migrate through 8 uM pore size modified Boyden Chambers (BD Biosciences, Bedford, MA) from upper chambers containing serum free medium to the lower chamber containing 10% FBS medium. After 4 hours, cells that migrated to the lower side of the membrane towards a higher FBS concentration gradient were fixed in 3.7% formaldehyde/PBS for 10 minutes, stained with 0.5% crystal violet and tabulated in triplicate. For clonogenicity assays, the cells were seeded in 6-well plates in low densities (5000 cells/well) and allowed to grow for 7 days in 400 μg/ml G-418 containing growth medium with a change of medium after 3 days. Cells were washed with PBS, fixed with 3.7% formaldehyde/PBS solution, stained with 0.5% crystal violet, and colonies with 10 +/− 2 cells were counted and averaged from 6 wells after performing the assay in quadruplicate.
Significance of observed differences in sample means was evaluated using independent samples t-tests or ANOVA where appropriate after ensuring normality of distribution (Shapiro-Wilk test) and equivalence of variance (Levene’s test). P-values less than 0.05 were considered to be significant in all cases. Analysis was performed using SPSS v.19 (IBM Corporation, Armonk, NY).