Establishing radio-resistant rectal cancer cell lines and cell culture condition
Seven rectal cancer cell lines (SNU-61, SNU-283, SNU-503, SNU-977, SNU-977R80Gy, SNU-1411, and SNU-1411R80Gy) were provided by the Korean Cell Line Bank (Seoul, Korea). Catalogues numbers of the cell lines were 00061, 00283, 00503, 00977, 00977-RAD, 01411, and 01411-RAD respectively. A total of 80 Gy of fractionated ionizing radiation were irradiated to three rectal cancer cell lines (SNU-61, SNU-283, and SNU-503) over 40 times by using Cesium-137 irradiator. The established radio-resistant rectal cancer cell lines were deposited by Korean Cell Line Bank. The catalogues numbers of the established radio-resistant rectal cell lines are 00061/R80GY, 00283/R80GY, and 00503/R80GY. All cell lines were cultured in RPMI1640 media with 10% FBS and penicillin (100 units/ml)-streptomycin (100μg/ml) (Thermo Fisher Scientific, CA, USA).
DNA fingerprinting analysis
A DNA fingerprinting analysis is used to authenticate each cell line. The genomic DNA from each cell line was amplified using the AmpFlSTR identifiler PCR amplification kit (Applied Biosystems, Foster City, CA, USA). A single PCR amplified 15 tetranucleotide repeat loci (CSF1PO, D2S1338, D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D19S433, D21S11, FGA, TH01, TPOX, and vWA) and Amelogen gender determining marker at loci containing highly polymorphic microsatellite markers. Amplified products were analyzed using an ABI 3730 genetic analyzer (Applied Biosystems). The STR profiles of established radio-resistance rectal cancer cell lines and their parental cell lines are listed in Additional file 1: Table S2.
3.0 × 105 cells were seeded in 3 ml of culture medium onto 6-well plates. Cells were maintained in humidified incubators at 37 °C in an atmosphere of 5% CO2 and 95% air for 24 h. Cells were exposed to 4 Gy of radiation using Cs-137 irradiator and stained with 0.4% trypan blue. The number of viable cell were counted using the Countess™ cell counting chamber slide (Invitrogen, Carlsbad, CA, USA) and Countess™ automated cell counter (Invitrogen) for 96 h in 24 h intervals. Every process was repeated three times for each cell line.
Cell viability assay
0.5 × 105 cells were seeded on 96 well plates with 0.5 ml of RPMI1640 media with 10% FBS and 1.1% penicillin for the cell viability assay. Meanwhile, 7.5 × 105 cells were simultaneously seeded on a T75 flask with 15 ml of RPMI1640 media at 10% FBS and 1.1% penicillin for Western Blotting. After 24 h of incubation at 37 °C in a 5% CO2 and 95% air atmosphere, cells were exposed to 12 Gy of Cs-137. In a time course manner (6, 24, 48, and 72 h after irradiation), 10 μl of EZ-Cytox solution (Daeil Lab, Seoul, Korea) was added to each well of 96 well plates. After 2 h of incubation at 37 °C, the optical density was measured at 450 nm by Multiskan™ GO Microplate Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). 1.0 × 105 cells were seeded on 96 well plates with 0.5 ml of RPMI1640 media at 10% FBS and 1.1% penicillin, which were irradiated with 0 and 4 Gy of Cs-137 after 24 h. 50 μl of MTT [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide] (Sigma-Aldrich co., St, Louis, MO, USA) solution diluted with PBS (2.5 mg/ml) was added to each well of the 96-well plates at the same time interval as the cell counting (0 and 96 h). After 4 h of incubation, the MTT solution was removed and 150 μl of DMSO (dimethyl sulfoxide) was added. The absorbance was measured by an ELISA reader (Molecular Devices Co., CA, USA) at 540 nm after being incubated for 15 min at room temperature.
Cell cycle analysis by FACS
7.5 × 105 cells were seeded on a T75 flask with 15 ml of RPMI1640 media with 10% FBS and 1.1% penicillin. After 24 h of incubation at 37 °C in an atmosphere of 5% CO2 and 95% air, cells were exposed to 4 Gy of Cs-137 and collected 96 h after irradiation. Collected cells were fixed with 70% ethanol and incubated at 4 °C for 24 h. After washing with cold DPBS, cells were stained with propidium iodide (PI) (100 μg/ml) (Sigma-Aldrich Co.) and RNase A (10 mg/ml) (Intron biotechnology, Gyeonggi, Korea) for 30 min on ice. Then, a fluorescence-activated cell sorter (FACS CantoII™, BD, NJ, USA) was used to analysis the cell cycle phases.
Colony forming assay
1.5 ml of 0.5% noble agar (BD Difco™, Franklin Lakes, NJ, USA) in RPMI1640 with 10% FBS was solidified at the bottom of each individual well in a 6-well plate. 3.5 × 103 cells in 500 μl of RPMI1640 media with 10% FBS and 1.1% penicillin were mixed with 500 μl of 0.4% agar and spread on the bottom agar. After 24 h of incubation at 37 °C in an atmosphere of 5% CO2 and 95% air, the plate was irradiated with 4 Gy of Cs-137. Every 5 d 300 μl of RPMI1640 media at 10% FBS and 1.1% penicillin was added to prevent desiccation of agar media. Three weeks later, the colonies were stained with 500 μl of 0.05% crystal violet and counted under a phase-contrast microscope. The number of colonies from triplicated wells was averaged.
Total RNA was extracted from induced radio-resistant and parental cell lines using TRIzol (Invitrogen, Carlsbad, CA, USA) and purified with the RNeasy Mini Kit (Qiagen, Hilden, Germany) according to manufacturer’s instructions. The RNA integrity was assessed by an Agilent 2100 Bioanalyzer (Agilent, Palo Alto, CA, USA). High quality RNA (RNA integrity number > 9.0) was used for the gene expression microarray analysis in which 100 ng of total RNA was processed for biotin labeled target preparation and hybridization to the Affymetrix Gene 1.0 ST array according to manufacturer’s instructions in order to perform the gene expression profiling experiments (Affymetrix, Inc., Santa Clara, CA, USA). After 16 h of hybridization at 45 °C and rotating at 60 rpm, the arrays were washed and stained on a GeneChip Fluidics Station (Affymetrix, Inc.) and scanned using the Gene Chip Scanner 3000 (Affymetrix, Inc.). The CEL intensity data extracted by GCOS (Gene Chip Operating Software) was used for the data analysis and raw data were processed using the Affymetrix® Expression Console software with the default RMA parameters.
RNA isolation and cDNA synthesis
Cells were collected by trypsinization and suspended in TRIzol (Invitrogen, Carlsbad, CA, USA). The total RNA was isolated with the RNeasy Mini Kit (Qiagen, Hilden, Germany) according to manufacturer’s instructions. For cDNA synthesis, QuantiTect Reverse Transcription Kit (Qiagen) is used. 1 μg of total RNA, 2 μl of gDNA Wipeout Buffer, and DEPC water filled up to 14 μl were mixed together and incubated at 42 °C for 2 min. The mixture was then blended with Quantiscript RT Buffer, RT Primer Mix, and Quantiscript® Reverse Transcriptase, and incubated at 42 °C for 45 min. The mixture was further incubated at 95 °C for 2 min and cooled down to room temperature.
Reverse transcriptase-PCR (RT-PCR)
Synthesized cDNA was diluted to 100 ng/μl using distilled water. 1 μl of 100 ng/μl cDNA was amplified in 14 μl of a PCR mixture that contained 1.5 μl of 10 x PCR buffer (with MgCl2), 0.5 μl of dNTP, 0.25 μl of forward primer (10 pmol/ul), 0.25 μl reverse primer (10 pmol/ul), 11.42 μl of distilled water, and 0.08 μl of i-Taq DNA polymerase (500 units) (Intron biotechnology, Gyeonggi, Korea). The primer sequences that were used in this study are listed in Additional file 1: Table S1. RT-PCR was performed using a programmable thermal cycler (PCR System 9700, Applied Biosystems; Foster City, CA, USA) and the RT-PCR products were fractionated on a 1.5% agarose gel containing ethidium bromide (EtBr).
Synthesized cDNA was diluted to 10 ng/μl and each primer concentration was optimized. 1 μl of cDNA was mixed with 5.0 μl of Master Mix (Applied Biosystems), distilled water, and optimized volume of each forward and reverse primer. The primer sequences were the same as the ones used in RT-PCR, as listed in Additional file 1: Table S1. A real-time PCR analysis was performed with the 7900HT Fast Real-Time PCR System (Life Technologies Co., Carlsbad, CA, USA) and the results were normalized to the housekeeping gene, β-actin, and the cycle threshold (Ct) values were extracted.
Protein isolation and western blotting
0.5 × 105 cells were seeded on 96 well plates with 0.5 ml of RPMI1640 media with 10% FBS and 1.1% penicillin for the cell viability assay while 7.5 × 105 cells were simultaneously seeded on a T75 flask with 15 ml of RPMI1640 media with 10% FBS and 1.1% penicillin for Western Blotting. Cells were harvested with a cell scraper after washing with cold PBS. Whole protein was extracted with EzRIPA buffer (ATTO Co., Tokyo, JAPAN) supplied with 1% protease inhibitor and 1% phosphatase inhibitor in accordance with the cell viability assay time frame. The volume of lysis buffer was adjusted to the number of cells collected in each vial. The protein concentration was determined by SMART™ micro BCA protein assay kit (Intron biotechnology, Gyeonggi, Korea). Equal amounts of protein were loaded on 4–15% Mini-PROTEAN TGX™ Precast Gels (BIO-RAD, Hercules, CA, USA) and blotted at 50 voles for 2 h. Proteins were then transferred to Trans-Blot Turbo™ Transfer Pack (BIO-RAD) using Trans-Blot Turbo™ Transfer System V1.02 machine (BIO-RAD) at 2.5 Amp and 25 Volt. The membrane was incubated in 2.5% skim milk containing 0.5% Tween 20 for an hour at room temperature. Primary antibodies against NDRG1 (abcam, Cambridge, United Kingdom) (1:5000), ERRFI1 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) (1:1000), PARP (BD Biosciences, San Jose, CA, USA) (1:1000), Caspase-3 (abcam, Cambridge, United Kingdom) (1:2000), and β-actin (Applied Biological Materials Inc., Richmond, BC, Canada) (1:5000) were diluted with 1.5% skim milk (BD Biosciences, CA, USA) containing 0.5% Tween 20 and introduced to the membrane. After 2 h at room temperature, Peroxidase conjugated mouse or rabbit IgG antibody (Jackson Immunoresearch, West Grove, PA, USA) (1:5000) was added as a secondary antibody. A chemiluminescent working solution, WESTZOL™ (Intron biotechnology), was decanted into the membrane, which was then exposed to Fuji RX film (Fujifilm, Tokyo, Japan) for 1–5 min.
Cells were seeded on chambered coverglass (Thermo Fisher Scientific, MA, USA) at densities of 3 × 103 to 2 × 104 cells/mL in culture media, according to diverse cell growth rates and desirable cell confluency. After 72 h, cells were washed with DPBS three times before being fixed and permeabilized using BD Perm/Wash™ (BD bioscience, CA, USA). After cells were washed with washing solution (BD bioscience, CA, USA), a PBS solution containing 2% FBS (GE Healthcare Life Sciences, Buckinghamshire, UK) was added. Primary antibodies that were used for Western Blotting were applied for immunofluorescent staining at dilution factors of NDRG1 (1:50), ERRFI1 (1:100), PARP (1:100), and Caspase-3 (1:100). 1 to 2 h after the primary antibody was applied, cells were washed with cold PBS containing 0.05% of tween 20. A conjugated secondary antibody (Thermo Fisher Scientific, MA, USA) was applied for 1 to 2 h in accordance with matched species. After cells were washed with cold PBS containing 0.05% of tween 20, distilled water with 1× DAPI and Rhodamine-conjugated Phalloidin (Sigma-Aldrich, MO, USA) was added for 20 min. Finally, the cells were washed with DPBS three times and viewed using a LSM800 Confocal Laser Scanning Microscope (Carl Zeiss, Oberkochen, Germany).
Knockdown of ERRFI1 expression by siRNA transfection
2.0 × 105 cells of SNU-503R80Gy with 1 ml of culture media were seeded in a 6-well plate and transfected with control siRNA and ERRFI1 siRNA (No#:1048480) (Bioneer, Alameda, CA, USA) with lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) at a final concentration of 40 nM siCONT and 40 nM siERRFI1 in Opti-MEM medium for 6 h. The media was then replaced with an equal volume of RPMI1640 (Gibco) without antibiotics. Consequently, the cells were collected for cell counting and cell viability assay for confirmation of the mRNA level.
Knockdown of NDRG1 expression by shRNA transduction
4 × 104 of 293FT cells with 10 ml of DMEM media supplied with 10% of FBS and 1% of penicillin streptomycin were seeded on 100 pi tissue culture. After 24 h of incubation, the culture media was changed to 10 ml of Opti-MEM, and ViraSafe™ Lentiviral Packaging System, Pantropic (CELL BIOLABS, INC., San Diego, CA, USA) with short hairpin RNA targeting NDRG1 (Sigma-Aldrich co., St, Louis, MO, USA) was treated with Lipofectamine 3000 (Invitrogen) in accordance with manufacturer’s protocol. After 48 h, the viral soup was harvested and filtered through a 0.45 μm pored filter (Sartorius Stedim Biotech SA, Göttingen, Germany). The resulting harvested viral soup was aliquoted into a 1.5 mL tube and kept at − 70 °C. 1 × 105 cells/ml of SNU-503R80Gy cells were seeded on 24 well tissue culture plates in 0.5 ml of RPMI1460 medium and incubated at 37 °C overnight. Viral transduction was performed using ViraDuctim™ (CELL BIOLABS, INC., San Diego, CA, USA) according to manufacturer’s protocol. The efficacy of shRNA on down-regulating NDRG1 was confirmed by Western Blot.
All acquired data in this study were analyzed by GraphPad Prism 5.0 and expressed as mean ± standard deviation. A comparison between the two cell lines (SNU-503 and SNU-503R80GY) was performed by a two-way variance analysis (two-way ANOVA) with the radiation time and dose as dependent variables.