Chemical reagents:
The primary antibodies used for the study, anti-p53, anti-phospho-Akt(S473), anti- FITC-conjugated anti-rabbit IgG were purchased from Genetex, CA, USA. An antibody against Aurora A was purchased from Biolegend. β-actin antibody was procured from abcam, Cambridge, UK. Anti-NFκB p50, p65, anti-Ki67, anti-PCNA, anti-MMP9 and anti-Oct 4 antibodies were obtained from Santa Cruz USA. Antibodies against phospho-Aurora(T288) and GADD45α were procured from Cell Signaling Technology, USA. Trypan Blue, PI (Propidium Iodide), DAPI (4′,6-diamidino-2-phenylindole), Aurora-A Inhibitor I or AKI and BSA (Bovine Serum Albumin) were purchased from Sigma-Aldrich, USA. All other reagents of analytical grade were procured locally.
Cell lines and cell culture:
Human cervical squamous carcinoma cell line SiHa was obtained from National Centre for Cell Science (NCCS), Pune INDIA. Subsequently a radioresistant subline SiHa/RR was developed by weekly incremental fractionated doses of irradiation. Cells were maintained in each irradiated dose for 5 passages and finally a colony was isolated at 40 Gy; designated as SiHa/RR. Both SiHa and SiHa/RR cells were maintained in MEM supplemented with 15% heat inactivated fetal bovine serum (FBS) and antibiotics (gentamycin 40 μg, penicillin 100 units, streptomycin 10μg/ml). Cells were maintained at 370C in a humidified CO2 incubator having 5% CO2/95% air.
Irradiation:
SiHa cells were initially, cultured to reach an approximate confluence of 50% in 25 cm2 cell culture flasks (Greiner Bio-One International). The culture medium was replenished with 2 ml of fresh complete MEM 15 min prior to administration of radiation dose. Flasks were then irradiated using X-Ray linear accelerator (Elekta). Medium of the post-irradiated cultured flask was replaced with 5 ml fresh medium before further incubation. Surviving cells were allowed to repopulate until reaching maximum confluency and then again subcultured into two new flasks after proper trypsinization. Upon attaining 50% confluency, these cells were subjected to another round of irradiation in the following week. The initial dose of irradiation being 2 Gy, successive weekly incremental doses of 0.5 Gy was administered for 15 weeks to achieve total cumulative dose of 40 Gy. A weekly maintenance dose of 2 Gy was continued to the cultured flask of finally isolated subline for sustaining adaptability of the cells. Further experiments were accomplished with exponentially growing cells after 48 h of the last maintenance irradiation.
Morphological observation and cell viability assay
SiHa and SiHa/RR cells were examined under Phase contrast microscope (Carl ZEISS) at the magnification of 400X. Images were captured and analyzed (Nucleus: Cytoplasm) using FIJI software. The alterations in the ratio were plotted graphically. Roughly 1x105 cells were seeded into a 25 cm2 flask (Greiner Bio One International) 48 h prior to the first irradiation. These cells were weekly exposed to previously mentioned doses of X-Ray. Cells were trypsinized 1 h after each irradiation and single suspended cells were counted using haemocytometer after staining with Trypan Blue for 15 min (Sigma). Unstained (viable) and stained (nonviable) cells were counted separately in the hemacytometer and percentage of viable cells were calculated as total number of viable cells / total number of cells X 100 and were calculated and plotted graphically for assessment.
Clonogenic viability assay:
Confirmation of acquired radioresistance was evaluated by the ability of colony formation upon challenging with acute radiation dose in both SiHa and SiHa/RR. Cells at a number of 1000 per well was seeded in 96-well plate. After 24 h of incubation, both SiHa and SiHa/RR cells were irradiated with an acute dose of 10Gy. Cellswere allowed to grow further till visible colonies were found. Colonies were subsequently stained with 0.5% crystal violet (Sigma, St. Louis, MO) and colonies of 50 cells or more were counted under microscope. Surviving fraction (SF) was calculated as percentage of number of colonies formed to total number of seeded cells. This set of experiment was furthermore repeated with pretreatment withaspecific Aurora Kinase Inhibitor I orAKI (0.01μM for 24 h) prior to exposure to acute 10Gy radiation dose.
Flow cytometric analysis:
Cell Cycle phase specific distribution of parental vs. radioresistant cells was evaluated using flow cytometry technique following standard laboratory protocol [14]. Both SiHa and SiHa/RR cells were treated with 10Gy of acute X- irradiation. After 6 h of treatment, 2x106 cells were suspended in cold PBS and centrifuged at 100 g for 8 min. Successive fixation of cells were done in 70% cold absolute ethanol for keeping under incubation in ice for 30 min. Cell pellets obtained after centrifugation and removal of ethanol were suspended in 1ml DNA binding solution (200 μg/ ml RNase + 50 μg/ml PI). Prior to analysis in Thermo Fisher Flow Cytometer, cell suspension in this condition was kept at dark for 30 min. Fluorescence was captured on BL2A channel and 10,000 cells with logarithmic amplification were counted. Percentage of AURKA expressed cells were determined flow-cytometrically by staining the acetone fixed SiHa and SiHa/RR cells with AURKA antibody tagged with Alexa Fluor 488 (Novus Biologicals) and Fluorescence was captured on BL2A channel.
Immunoblotting technique:
Protein expression was determined by standard laboratory technique of immunoblotting [15]. Briefly, 6 h post irradiated (10Gy) SiHa/RR as well as parental radiosensitive SiHa cells were harvested and cell lysates were prepared using lysis buffer. Additionally in another set, SiHa/RR cells were pretreated with AKI (24h) followed by 10 Gy irradiation with an incubation for 6 h followed by cell lysates preparation. Proteins were quantitated by Bradford colorimetric assay. Electrophoresis (Bio-Rad apparatus) of equal amounts of total proteins were performed on SDS-polyacrylamide gel using electrophoresis buffer (Tris: 25 mM, glycine: 192 mM, SDS: 20%) and electro-transferred to nitrocellulose membranes using transfer buffer (Tris: 250 mM, glycine: 192 mM, methanol: 10%). Membranes were blocked in 5% BSA; thereafter incubated overnight with respective primary antibodies at 40C. Following incubation, membranes were washed with TBST (Tris Buffered Saline with Tween20) thrice and further incubated with alkaline phosphatase-conjugated anti-mouse IgG or anti-rabbit IgG (1:1000 dilutions in TBS). TBST was used for subsequent washing of membranes for removal of nonspecific binding of secondary antibodies. Proteins were thereafter visualized upon addition of BCIP/NBT in the form of colorimetric bands. The comparative expression profiles of Ki-67, PCNA, AURKA, p53, Gadd45α HIF1 α, NFκB p50, NFκB p65, Akt, phospho Akt, MMP, Oct4 was assessed using β-actin as loading control.
In silico clustering analysis:
String Database was used to study the interactions between the proteins which were found to be overexpressed in SiHa/RR cells based on the formula Network analysis = Network Enrichment Analysis + Network Topological Analysis.
In Vitro Scratch (migratory) assay:
Alteration of cell motility was determined using in vitro scratch or migratory assay. A scratch was drawn in a straight line over the monolayer of the confluent culture using 200µl sterile pipette tip. The treatment conditions were a). Untreated SiHa cells, b). 6 h post irradiated cells (10Gy acute shot) and c). Cells pretreated with AKI (0.01 μM) for 24 h followed by 10Gy X-irradiation and subsequent incubation for 6 h. The image of the scratch width was captured under a phase-contrast microscope, immediately after treatment (0 h) and again after 36 h of incubation. Percentage of wound healing area was determined by calculating the reduction of the scratch-width (width of the space devoid of migrating cells) after the indicated incubation time and expressed as percentage of wound closure. Scratch area was calculated using FIJI software.
3D sphere formation Assay:
3D sphere forming assay was carried out using hanging drop method as described by Foty with slight modifications [16]. Treatment conditions were similar to that of wound healing assay. Following completion of treatment, cells were trypsinized and resuspended in the medium. The lid of a 60 mm tissue culture plate was removed and inverted to deposit 10 μl drops onto the bottom of it using 20 μl pipett (up to 20 drops were placed in well maintained distance from each drop). The lower plate was filled with 5 ml of sterile PBS to use it as a hydration chamber. The PBS-filled chamber was covered by the lid containing cell droplets. Such plates were kept for incubation at 370C/5% CO2 with95% humidity till visible cell sheets or aggregates were found on the lid. After microscopical confirmation of conspicuous cell sheets, those were transferred to 25 cm2 flasks filled with 3 ml complete medium and were placed in shaker incubator at 370C and 5% CO2 till the development of spheroids. Microscopic observation and capturing of image were performed at the 100X magnification using a phase contrast microscope (Zeiss). Further measurement of sphere area was performed using FIJI software.
Apoptotic assay:
To examine the characteristic morphological features of apoptosis, bothSiHa and SiHa/RR cells were exposed to acute shot (10 Gy) of irradiation. Cells were harvested, 6 h after irradiation, washed with PBS and centrifuged and to the pellet PI (Sigma-Aldrich) was added (final: 50μg/ml). AKI pretreated cells (as described in earlier experimentations) were also stained with PI. Cells were then incubated in the dark at RT for 10 min. Both treated and untreated cells were smeared in the glass slide, covered with cover slip, and observed under the fluorescent microscope. Characteristic apoptotic cells were examined under a fluorescence microscope at PI specific filter (Leica).
Ectopic overexpression of AURKA using plasmid DNA:
Confluent SiHa cells were added with specific plasmid with the modified pcDNA5/FRT/TO vector containing a single amino-terminal Myc tag and the human wild type AURKA transgene (Plasmid #59804; pcDNA5 FRT TO Myc ArA WT, AddGene) and lipofectamine to Opti-MEM aliquots (250 μl each) and incubated for 5 min at room temperature. Thereafter, plasmid/Opti-MEM and the Lipofectamine 3000/Opti-MEM was mixed and allowed to incubate for 20 mins at room temperature. Plasmid containing medium was added to cells in culture. Finally, expression was checked by western blotting and immunofluorescence assay.
Statistical Analysis:
Comparative analysis of differences between groups was carried out using Student’s t test in the GraphPad Prism 5.0 software package (GraphPad Software, Inc., La Jolla, CA, USA). p<0.001 was considered the minimal level of significance. Data or values were obtained from at least three independent experiments and expressed as Mean ± SD.