Cell lines, Antibodies, and Reagents
FaDu and PA317 LXSN 16E6/E7 cell lines were procured from American Type Culture Collection (ATCC, Manassas, VA) and grown in DMEM media containing 10% Fetal Bovine Serum (Fisher) and 1% penicillin-streptomycin solution (penicillin, 10,000 U; streptomycin, 10 mg/mL; both from Fisher) at 37°C in a 5% CO2 incubator. The cell lines FaDu and PA317 were screened for mycoplasma by the manufacturer, ATCC, using both the Hoechst DNA stain and direct culture method. The antibiotics amount in the cell culture medium is recommended by ATCC and is considered inconsequential for cellular responses. The FaDu line was established in 1968 from a punch biopsy of a hypopharyngeal tumor. PA317 LXSN 16E6/E7 is a packaging cell line developed by transfection of the retrovirus vector pLXSN16E6/E7 into the Psi-2 ecotropic packaging cell line. The pLXSN16E6E7 vector contains the human papilloma virus (HPV) type 16 E6 and E7 genes under control of the Moloney murine leukemia virus (MoMuLV) promoter-enhancer sequences. The vector also contains a gene encoding resistance to neomycin transcribed from the SV40 promoter. This line produces the amphotropic retrovirus LXSN16E6/E7 encoding the HPV16 E6 and E7 open reading frames, which have been used to stably infect and immortalize many cell types. Cervical carcinoma HPV16-positive CasKi cell line (ATCC) was grown as in [6]. Murine anti-HPV16-E6, C1P5 antibody (IgG1 isotype) was obtained from Abcam (Boston, MA). Proteasome inhibitor MG-132 was purchased from Calbiochem (San Diego, CA) and BD Matrigel Basement Membrane Matrix - from BD Biosciences (Rockville, MD). The beta-emitter 188Re with a half life of 16.9 hours was produced from beta decay of 188-Tungsten parent (half life 69 days) using a 188W/188Re generator (Oak Ridge National Laboratory, Oak Ridge, TN). After 188Re was eluted in the form of sodium perrhenate, the C1P5 mAb was labeled "directly" with 188Re through binding of reduced 188Re to the generated -SH groups on the mAb as previously described [8–10].
Transfection of FaDu cells with HPV16 Oncogenes E6 and E7
Transfection of the FaDu cell line with the HPV16 oncogenes E6 and E7 of PA317 LXSN16E6/E7 was performed by growing PA317 LXSN16E6/E7 cells in complete media until ~85% confluence. The media was changed and 16 hours after incubation with fresh media, the media containing the virus was removed and passed through a 0.4 μm low protein binding filter to remove cells and debris. The supernatant containing the virus was kept for further use. The FaDu cells were incubated under the same conditions until ~60% confluence. One mL of viral supernatant was combined with 3 mL of serum-free DMEM containing 4 μg/mL hexadimethrin bromide (polybrene) (Sigma) and overlaid onto the FaDu cells in 75 cm2 flasks. After incubation for 3 hours, another 5 mL of serum-free DMEM containing 4 μg/mL polybrene was added to the flasks and incubated for another 4 hours. The media/viral supernatant mixture was then removed and 25 mL of complete DMEM was added to each flask, which were incubated for another 48 hours. Selection of transfected cells was done using the neomycin analogue, G418 (1 mg/mL) (Fisher), for 7 days. The surviving cells were then cultured with complete DMEM containing 200 μg/mL G418 for the duration of the project. Pure colonies were established by serially diluting the surviving cells in a 96-well plate. Wells containing only one cell were grown and eventually transferred to 75 cm2 flasks for further propagation. The growth of the transfected cells was compared to that of FaDu cells for 6 days and doubling times for both cell lines were calculated using the following formula:
DNA Extraction and PCR
Polymerase Chain Reaction (PCR) was used to determine the presence of oncogenes E6 and E7 in the transfected cell lines. DNA from the colony purified line was extracted and purified using the DNeasy Blood & Tissue Kit obtained from Invitrogen. The concentration of DNA was measured spectrophotometrically at 260 nm wavelength using a Spectra Max 250 96-well plate reader. The following oligonucleotides (Invitrogen) were used: E6F1 5'-ATGTTTCAGGACCCACAGGA-3'; E6R1 5'-GGTTTCTCTACGTGTTCTTGA-3'; E7R1 5'-TCCAGCTGGACCATCTATTTC-3'. The E6 and E7 oncogenes overlap on the vector thus allowing for two products - E6 alone and E6 + E7. Platinum PCR SuperMix High Fidelity from Invitrogen was added to each PCR reaction tube along with 150 ng of genomic DNA, and 3.0 μL of primer (300 nM). The tubes were incubated initially at 94°C for 1 min to denature the template and to activate the enzyme followed by 40 temperature cycles of: 95°C for 30 seconds, 50°C for 40 seconds, 68°C for 40 seconds. DNA from CasKi cells which have high number (~ 600) of HPV16 copies per cell was used as positive control.
Western Blotting
Western blotting was performed to establish the presence of E6 oncoproteins in the transfected FaDu cells as well as to estimate the level of E6 expression in this cell line in comparison with cervical cancer cell line CasKi. Cell pellets were suspended in the lysis buffer (4% SDS, 20% glycerol, 0.5 M TrisHCl (pH 6.8), 0.002% bromophenol blue and 10% b-mercaptoethanol). Protein samples were boiled in water for 5 min before running SDS-PAGE. Twenty five to thirty μl of protein solution was loaded into each well of 12% pre-cast SDS-PAGE gel (Bio-Rad). SDS-PAGE was used to monitor the relative protein amounts in the samples. All samples were normalized with a tubulin control. Twelve percent SDS-PAGE gel was used to separate proteins, and electrophoresis was performed using Mini-Protean® 3 Cell system (Bio-Rad). After electrophoresis, the gel was transferred into the PVDF transfer buffer (25 mM Tris, 190 mM glycine and 2.5% (v/v) methanol) for 5 min. Then, proteins were transferred from the gel to the Immun-Blot™ PVDF membrane (Bio-Rad) on Semi-dry Electrophoretic Transfer Cell (Bio-Rad) at 15 V for 17 min. The membrane was soaked in the blocking solution (5% non-fat dry milk in the 0.1% TBST) with gentle shaking for one hour. The membrane was incubated in TBST blocking solution (0.1% Tween-20, 25 mM TrisHCl (pH 7.6), 500 mM NaCl, and 5% non-fat dry milk) containing 1:2000 diluted anti-HPV16 E6 C1P5 mAb at 4°C overnight. After several washes with TBST, the membrane was hybridized by the secondary antibody conjugated with horseradish peroxidase (Rabbit polyclonal to mouse IgG (HRP)) in TBST blocking solution with a 1:10,000 dilution. After three washes with TBST, the membrane was incubated with Rodeo ECL detection reagents 1 and 2 (USB) for 5 min, and then exposed to CL-XPosure™ film (Pierce). The film was developed as per manufacturer's instructions. For estimation of E6 level in HPV transfected FaDu cells - the western blotts of this and of CasKi cell lines were subjected to ImageJ intensity quantification.
Tumor Model
All animal studies were carried out in accordance with the guidelines of the Institute for Animal Studies at the Albert Einstein College of Medicine. 6-8 weeks-old athymic Nu/Nu CD1 nude mice were purchased from Charles River Laboratories. Ten million HPV transfected FaDu cells (3-4 passaging of cells) were mixed with 80% Matrigel and injected subcutaneously into the right flank of each mouse. The size of the tumors was measured every 3 days with calipers (Pro-max Sylvac System IP67, Fowler Tools and Instruments, Boston, MA) in three dimensions and the tumor volume was calculated as a product of three dimensions divided by 2. Electronic calipers help to ensure the precision of measurement and allow the immediate download of the measurements into the Excel file for tumor volume calculations, groups randomization etc.
MG132 Treatment of Transfected FaDu Cells in vitro and in vivo
To study the response of HPV-transfected FaDu cells to MG-132 (Z-LLL-CHO, MW = 457.6) which is a potent, reversible and cell-permeable proteasome inhibitor (Ki = 4 nM) - the original and transfected FaDu cells were harvested and transferred into 4 sterile test tubes with complete media. Each tube contained 0.5 - 1 ml cell culture (cell concentration was ~106 cells/ml), and cells were allowed to grow at 37°C in 5% CO2 incubator overnight. Then, MG-132 solution was added to the tubes for the final concentrations of 0, 5, 10 or 25 μg/ml. The cells were incubated under the same conditions for another 3 hours. Finally, the cells were harvested by centrifugation, clarified by washing with PBS and processed for western blot as described above. For in vivo investigation of the MG-132 influence on the level of E6 expression in HPV-transfected FaDu tumors, 3 mice carrying tumors were injected intraperitoneally (IP) with 20 μg MG-132 and another group of 3 transfected tumor-bearing mice was used as control. Three hours later, all mice in both groups were sacrificed, their tumors removed, homogenized on ice and processed for western blot as described above.
RIT of Transfected FaDu Tumors
To investigate the utility of the transfected FaDu cell line as a basis for an animal model for drug development, we conducted a proof of principle RIT study targeting E6 oncoprotein with 188Re-labeled C1P5 mAb. The mice with transfected FaDu tumors measuring 0.5-0.7 cm in diameter were randomized into groups of five. The groups were treated IP with either: 200 μCi 188Re-C1P5 mAb, matching amount (6 μg) of unlabeled ("cold") C1P5 mAb; or left untreated. The mice were observed for tumor growth and survival for 15 days. The experiment was performed twice.
Statistical Analysis
Student t-test was performed to compare the doubling times for FaDu and transfected cell lines. The sample sizes in the animal experiments were pre-planned. The differences between the tumor sizes for differently treated groups in the RIT studies were analyzed by non-parametric Mann-Whitney test using Prism software (GraphPad, San Diego, CA). The differences were considered statistically significant when p-values were < 0.05.