Cell lines and cell culture
W12 cell line was derived from a low grade cervical lesion by Stanley MA et al. 1989, and is unique among HPV16-containing cell lines in carrying its HPV 16 genome as a multicopy episome
. We made use of a pair of isogenic cell lines, W12 and S12 to compare difference of survival after irradiation. W12 cells contain episomal HPV 16 genomes, whereas S12 cells, which derived from the W12 line, contain HPV DNA as integrated copies
W12 cells were cultured with lethally irradiated Swiss 3T3 feeder cells and in medium consisting a mix of one-quarter Dulbecco’s modified eagle’s medium (Gibco) and three-quarters Ham F-12 medium (Gibco) containing 5% fetal calf serum, penicillin, streptomycin, an supplements (all from Sigma) as follows: 8.4.ng of cholera toxin/ml, 5 μg of insulin/ml, 24.3μg of adenine/ml, 0.5 μg of hydrocortisone/ml and 10 ng of epithelial growth factor per ml. Cells were split when reached 80% confluence. S12 cells were obtained by collecting surviving W12 cells cultured without feeder layer support.
E2-gene specific PCR
HPV16-positive cells were tested for an intact E2 gene in three separate amplification reactions which allows amplifying three amplicons of different length determining integrity of E2-gene
. The procedure and primer sequences used were as described in the same reference.
Clonogenic growth assay and irradiation
Clonogenic survival was analyzed by using 96-well in vitro test as followed: 1-100 cells per well were seed. The plates were examined with an inverted phase contrast microscope at intervals of 7, 10, 14 days. A well was considered positive when a colony in it reached a size of 50 cells or more. Cells were fixed with 70% for ten minutes prior staining with 0.1% methylene–blue. After staining weels were washed with destilled water. Plating efficiency (PE) was calculated using poisson statistics according to formula PE = -ln (neg wells/total wells)/ number of cells plated per well
. In radiation experiments fraction of survival was determined by dividing number of positive wells/plate/number of cells plated per well in irradiation group by number of positive wells/plate/number of cells plated per well in control plates. At least three plates were used for each group.
Cells were irradiated with singles doses of 0 Gy, 1 Gy, 2 Gy, 3 Gy, 4 Gy, 5 Gy and 7 Gy. In such experiments, an increasing number of cells plated for each increment in radiation dose. Therefore, effect of cell number per well on plating efficiency was evaluated. Plating densities of 1-10 cell/weel were tested. Although number of wells with colonies increased with higher cell density, plating efficiency was not effected by number of cells. When 10 cells/well were used all wells in this set of experiments contained colonies.
Survival curves were based on number of positive wells or colonies in each irradiated group as a fraction of that in control group. Survival curves where calculated using Sigma Plot 8.0. At least three experiments where performed for each dose point.
Cell cycle analyses
Cell cycle analyses were performed after 0 h, 6 h, 12 h, 24 h, 48 h and 7 days irradiation with 2 Gy and 7 Gy using flow cytometry using Propidium-Iodid (PI)-staining as described elsewhere
. Data were collected by using FACScan flow cytometry, and results were analyzed by using cellquest software (both from Becton Dickenson). For each sample, 10000 events were collected, and aggregated cells were gated out.
Intracellular cytokine staining: pRb and p53
The retinoblastoma gene encodes a nuclear phosphoprotein which is expressed in most normal cells and acts as a tumor suppressor. An underphophorylated form of Rb binds to viral oncogene HPV-E7
. Clone G3-245 recognizes an epitope between amino acids 300-380 of the human retinoblastoma protein (pp110-114 Rb).
Wildtype p53 formes specific complexes with several viral oncogenes including HPV-E6 and plays a role as checkpoint protein for DNA damage during G1/S-phase of cell cycle
. Clone G59-12 recognize mutant and wild type human, mouse and rat p53 suppressor protein.
The G3-245 or G59-12 and MOPC-21 FITC (a mouse IgG1 isotype control) conjugates are matched and F/P ratios determined experimentally by flow cytometric analysis.
Details of the procedure are described as follows:
Ethanol fixated cells were washed two times in cold PBS then resuspended in Fixation/Permialisation solution Perm/WashTM BD (1x106 cell/ml) for 30 min at 4°C and pelleted by centrifugation. Afterwards buffer was removed and cells were washed two times in fresh Perm/WashTM BD buffer. Thoroughly resuspended cells were subjected to intracellular cytokine staining by incubating in 100μl Perm/WashTM BD buffer containing 20μl of Fluorochrome-conjugated antibody Rb-ak (FITC Mouse Anti-Human Retinoblastoma Anti-Body from Becton Dickinson, BD-Set:# 556538 Clone G3-245) for 24h at 4°C temperature in the dark. After washing with Perm/WashTM BD cells were pelleted and resuspended cells in 0,5 ml Perm/WashTM BD for flow cytometric analysis. The same procedure was performed for p53 staining using 20μl of Fluorochrome-conjugated antibody p53-ak (FITC Mouse Anti-Human p53 Anti-Body from Becton Dickinson, Clone G59-12).
Flow cytometric analysis
Stained cells were analyzed using FACSCan flow cytometry (BD) equipped with a air-cooled 488 nm argon-ion laser. Data acquisition and analysis were performed using FACSComp and CELLQuest (version 3.4) software. A total event of 10 000 cells were acquired for each sample. Data were expressed as geometric mean fluorescence intensity and as ratio between fluorescence emission of sample cells and that of isotypic control (P/N ratio; positive/negative). In each case negative control were cells treated as described above without Rb-ak staining or p53-ak staining. Isotypic control were cells treated with isotype–matched control of irrelevant specificity from FITC Mouse IgG1 Isotype control (BD-Set# 556538) instead of Rb-ak staining or p53-ak staining. Analyses were performed after 0 h and 24 h irradiation with 2 Gy and 7 Gy.