HPV16-positive SiHa and CaSki cervical carcinoma cells were supplied by China Center for Type Culture Collection (CCTCC). The following cell lines used in this project were from our laboratory: HPV18-positive HeLa cervical carcinoma cells, CNE nasopharyngeal carcinoma cells, HaCat human keratinocyte cells, COS7 (African green monkey SV40-transformed kidney fibroblast cell line), 293T (human kidney epithelial cells), and NIH/3T3 mouse embryonic fibroblast cells. All cells were maintained in a humidified atmosphere of 5% CO2, Dulbecco's modified Eagle's medium (DMEM, pH 7.2), supplemented with 10% fetal calf serum (FCS).
Antibodies and Reagents
Mouse anti-HPV16E7, mouse anti-p107 (SD107), mouse anti-cyclin E1, mouse anti-cyclin A1 and mouse anti-cyclin D1 monoclonal antibodies were supplied by Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Rabbit monoclonal to pRb antibody was supplied by Abcam Ltd. (Hong Kong, P.R. China). Mouse monoclonal to p130/Rb2 antibody was supplied from NeoMarkers For Lab Vision Corporation (Fremont, CA, USA). pRb Antibody Sampler Kit, including Phospho-pRb (Ser780), Phospho-pRb (Ser795) and Phospho-pRb (Ser807/811) antibodies and pRb (4H1) Mouse antibody, was supplied by Cell Signaling Technology, Inc. (Danvers, MA, USA). HRP-coupled mouse anti-M13 antibody, HRP-conjugated goat anti-mouse IgG and HRP-conjugated goat anti-rabbit IgG were supplied by Amersham Biosciences (Piscataway, NJ, USA) and KPL (Gaithersbug, MD, USA), respectively. Mouse anti-HPV18E7 serum was prepared in our laboratory. TMB substrate kit was obtained from eBioscience Inc. (San Diego, CA, USA). Ni-NTA agarose beads (His-band Resin) and protein G agarose beads were supplied by Amersham Biosciences (Little Chalfont, UK). The Ph.D.-C7C™ phage display peptide library kit was purchased from New England Biolabs Inc. (Cambridge, MA, USA). DMEM, trypsin, and TRIzol reagent were supplied by Invitrogen (Carlsbad, CA, USA). Fetal calf serums (FCS) were purchased from Sijiqing (Hangzhou, P.R. China). 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide) (MTT) and dimethylsulfoxide (DMSO) were supplied by Sigma Chemical Co., (Aaint Louis, MI, USA). 5-Bromo-2′-deoxyuridine(BrdU) and Monoclonal Anti-BrdU antibody produced in mouse were bought from Sigma-Aldrich, Inc. (Aaint Louis, MO, USA). Other chemicals were of analytical grade and obtained from local commercial resources.
Plasmids and Reporter Constructs
The primers used in plasmid constructs of this study are listed in . HPV16E7-pEYFP plasmid was constructed by sub-cloning the HPV16E7 gene into the Eco
RI and Bam
HI sites of pEYFP-N1. The HPV16E7 gene was obtained by PCR from HPV16E7- pET28a (+) plasmid, which was maintained by our laboratory, using primers pE7N1f and pE7N1b. The pRb gene was obtained by nest PCR from HeLa cDNA using pRb1, pRb2, pRb3, pRb4 as primers. The PCR product was then inserted into the Hind
III and Xho
I sites of pcDNA3.1-His/V5-Topo. pA3-Luc-Cyclin D1 luciferase reporter plasmid containing the cyclin D1 promoter sequence −1745 to +134 was maintained by our laboratory. Both pCCNE1-Luc and pCCNA1-Luc were constructed by nest PCR to amplify fragments of cyclins E1 and A promoter sequences (cyclin E1: +3 to +594; cyclin A: −284 to +254) which include E2F binding sites 
from HeLa cDNA, respectively. Primers used in pCCNE1-Luc construction were P(CCE1), P(CCE2), P(CCE3), and P(CCE4), while primers P(CCA1), P(CCA2), P(CCA3), and P(CCA4) were used in PCCNA1-Luc construction. The PCR products were then cloned into the Kpn
1 and Bgl
II sites of the pGL3-enhancer (Promega, Madison, WI, USA) luciferase reporter plasmid. The double mutants of E2F sites in the pCCNE1, which changed TGTCCCGC
at position +7 to TGTCATGC
and changed GCGCGCAA
at position +497 to GCGATCAA
, were performed by site-directed mutagenesis using the megaprimer-PCR method as described previously 
. Luciferase reporter plasmid pMCCNE1-Luc, was made by sub-cloning of the corresponding PCR fragments into pGL3-enhancer, respectively. Primers P(pGL31),P(CCME1), P(CCE3), P(CCE4) and P(CCME2) were used in the megaprimer-PCR processes. The E2F binding sites muted cyclin A promoter luciferase reporter plasmid, pMCCNA1-Luc, in which the E2F binding sites TTTGGCTC
at position +31 and TTTGGGCG
at position +165 were changed into TTTGATTC
, respectively, was generated form pCCNA1-Luc as above, using P(pGL31), P(CCMA1), P(CCA3), P(CCA4), and P(CCMA2) as primers. The mutations were confirmed with DNA sequencing by Shanghai Sangon Biological Engineering Technology and Service Co., Ltd. (Shanghai, P.R. China).
Primer sequences used in plasmid constructs.
Protein Expression and Purification
The HPV16 E7 gene was amplified by PCR and cloned into the BamHI/EcoRI sites of the pET28a (+) vector (maintained by our lab). The pET28a (+)-HPV16E7 clone was then transformed into Escherichia coli BL21. Expression of His-E7 was induced by 1 mmol/L IPTG; the protein was extracted in denaturing conditions according to the Qiagen protocol and its purification carried out by immobilized-metal affinity chromatography with Ni-NTA agarose beads following the manufacturer's instructions (Amersham Biosciences, Little Chalfont, UK).
Phage Display Screening
HPV16E7 protein [100 µg/ml in 0.1 M NaHCO3 (pH 8.6) with 5 mg/ml BSA] was immobilized in 96-well microtiter plates (Corning, NY, USA) and incubated overnight at 4°C. The following day, the wells were blocked by incubation for 2 h at 37°C with 3% BSA and rinsed five times with TBST [Tris·Cl (pH 7.5) 50 mmol/L, NaCl 150 mmol/L, and 0.1% Tween 20]. The plates were subjected to panning for 1 h at room temperature with the phage particles, followed by washing three times for 15 min with TBS [Tris·Cl (pH 7.5) 50 mmol/L, NaCl 150 mmol/L]. Thereafter, the bound phages were eluted for 1 h at room temperature with 1 mg/ml HPV16E7 protein in 0.2 M Glycine-HCl (pH 2.2), followed by neutralization with 1.0 M Tris-HCl (pH 9.1). The recovered phages were amplified in Luria-Bertani plates containing stationary phase ER2537 cells. Amplified phages were screened in additional rounds and then plated to obtain isolated clones.
To characterize the individual clones' binding to HPV16E7, plaques were picked at random, amplified, and tested for HPV16E7 binding ability by ELISA using HRP conjugated anti-M13 antibody (Amersham Biosciences, Piscataway, NJ, USA), followed by TMB substrate coloration (eBioscience, San Diego, CA, USA) for colorimetric evaluation. The absorbance value was measured at 450 nm (A450) on a microplate reader. The PBS was immobilized to the plates as negative control. The clone was identified as positive if the A450 value of the sample well was three times higher than that of the negative control well.
Analysis of Amino Acid in Phage Clones and Peptide Synthesis
The nucleotide sequences coding for random peptides displayed on the positive-phage coat proteins were determined by Shanghai Sangon Biological Engineering Technology and Service Co., Ltd. (Shanghai, P.R. China). The corresponding peptides were chemically synthesized with or without an N-terminal fluorescein label (Chinese Peptide Company, Hanzhou, P.R. China) for further study. The peptide with the same amount of amino acid, but having random sequences, was synthesized as a negative control peptide. Crude peptides were purified by HPLC and checked by mass spectrometry. Except otherwise noted, peptides were dissolved in DMSO to obtain 10 mmol/L solutions, supplemented with equimolar DTT, and stored at −20°C.
Competitive ELISA Assay
The specificity and affinity of the positive clones and synthetic peptides were determined by competitive ELISA. 400 ng/well HPV16 E7 protein was coated overnight in microtiter wells at 4°C. The positive clones or peptides at different concentrations mixed with HPV16E7 mAb were incubated with the coated E7 protein in microtiter wells for 3 h. The mixture at the same concentration of the library phage and HPV16E7 mAb was set as negative control. The reaction was revealed by incubation with the HRP-conjugated goat anti-mouse antibody (KPL, Gaithrsbug, MD, USA) as described above. HPV16E7 mAb alone at the same concentration, incubated with the coated E7 protein, gave benchmark absorption. The inhibitory ratio was calculated by the following formula: Inhibitory ratio
100%−(A450 of mixture well/A450 of mAb alone well)×100%.
Peptide Internalization and Visualization
The 5-FAM labeled peptides were used to monitor the cell uptake and intracellular location of the peptide. SiHa cells were seeded on the coverslips in 12-well plates (1×105/well) and cultured overnight. The DMEM was replaced with a fresh medium, supplemented with 80 µM 5-FAM labeled peptide. Cells were washed three times with a PBS buffer after 8 h and 24 h of incubation, respectively, and then observed under a fluorescence microscope Leica DMI 6000B. Images were captured with Leica AF6500 software (Leica Mikrosysteme Vertrieb GmbH, Wetzlar, Germany). The percentage of cells up-taking F-pep was analyzed by flow cytometry sorting.
Cell Viability Assay
Cell viability was determined with an MTT assay. Briefly, the cells were seeded into 96-well plates at a density of 4–5×103 cells/well. The cells were allowed to recover for 24 h before the medium was replaced with DMEM supplemented with peptides at different concentrations. The solvent of the peptides was set as the control. The peptide-supplemented medium was replaced daily and the respective concentrations were maintained. The cells were subjected to an MTT cell proliferation assay at different time points. The plates were incubated at 37°C for 3 h with the addition of 20 µl/well MTT (5 mg/ml). Untransformed MTT in the solution was removed by aspiration. The formazan product was dissolved in DMSO, and plates were shaken vigorously at room temperature for 10–15 min to ensure complete solubilization. The optical density of the formazan solutions was measured by microplate reader at 540 nm.
Cells were seeded in DMEM at 1×103 cells/well density in 12-well plates and allowed to attach to the substrate overnight. Thereafter, cells were treated with 0 µM, 16 µM, 32 µM, 80 µM, and 160 µM peptide in DMEM, and the medium was changed every three days. The plates were observed daily for colony formation. After 8 (for HeLa cells) or 14 (for SiHa and CaSki cells) days of treatment, the cultures were washed three times with PBS and stained with crystal violet in ethanol for 15 min. The colonies were then counted and photographed with the Alphalmager® EP image acquisition system (Alpha Innotech, Santa Clara, CA, USA).
Cell cycle distribution was determined by flow cytometry DNA analysis. SiHa cells (2×105/well) were allowed to attach to the substrate overnight and then treated with or without 32 µM and 80 µM Pep-7 in full-culture medium. After 24 h, cells were harvested and counterstained with PI (Invitrogen, Carlsbad, CA, USA) for flow cytometry according to the manufacturer's instructions. Briefly, cells were trypsinized and resuspended in 1 ml PBS. Cell suspension was dropped slowly into 4 ml anhydrous ethanol pre-cooled at −20°C and mixed. After standing at −20°C for more than 15 min, the ethanol was removed by centrifugation, after which 5 ml PBS was added and mixed, and cells were re-hydrated at room temperature for 15 min. PBS was then removed by centrifugation. Cells were stained with 2 µg/ml propidium iodide in 1 ml dilution buffer (100 mmol/L Tris, pH 7.4, 150 mmol/L NaCl, 1 mmol/L CaCl2, 0.5 mmol/L MgCl2, 0.1% Nonidet P-40, 100 µg/ml RNAase) at room temperature for 15 min and subjected to flow cytometry analysis. Analysis was performed on an Epics-XL flow cytometer (Beckman Coulter, Inc. CA, USA) with Modfit 3.0 software (Verity Software House, Topsham, ME, USA).
Western blotting was carried out as described previously 
. The cells were seeded into 6-well plates, allowed to attach to the substrate overnight, and then treated with or without 80 µM Pep-7. After another 48 h of treatment, the cells were lysed in lysis buffer (Beyotime Institute of Biotechnology, Jiangshu, China) containing PMSF (Sigma Chemical Co., St. Louis, MI, USA) for 30 min at 4°C. The lysate was then centrifuged for 20 min at 13,000 rpm and 4°C. The proteins were then separated through SDS-PAGE and transferred onto the PVDF membrane (Immobion®-P Transfer Membrane, Millipore Corp., Billerica, MA, USA). Membranes were blocked in a Tris-buffered saline with 0.1% Tween-20 (TBS-T) solution with 5% nonfat dry milk and incubated overnight with primary antibodies at 4°C. The immunoreactive signals were detected by HRP-conjugated secondary antibodies (KPL, Gaithersburg, MD, USA) followed by SuperSignal® West Pico Chemiluminescent Substrate (Thermo Scientific, Milford, MA, USA.). Anti-β-actin antibodies were used to control protein loading. For the in vivo
studies, tumors were harvested, and the cell lysates were prepared and transferred to a clean Eppendorf tube and centrifuged for 30 min at 14,000 rpm. The supernate was probed by western blotting, as described above.
For pull-down analysis, the purified His-HPV E7 protein (200 µg/ml×50 µl) was preincubated with 100 µl His-band Resin (Amersham Biosciences, Little Chalfont, UK) for 4 h at 4°C. The beads were then washed four times with lysis buffer and divided into two equal portions. One half was added with the supernate of the 293T cell lysate with 300 µg total proteins and 10 µl (8 mmol/L) Pep-7, while an equal amount of cell lysate supernate and 10 µl PBS as control was added to the other half. The mixtures were incubated overnight at 4°C under continuous agitation. Beads were subsequently washed four times with a lysis buffer. Bound proteins were eluted by boiling in Laemmli sample buffer and analyzed by Western blot.
For Co-IP, Cos-7 cells seeded in 100 mm plates were co-transfected with HPV16E7-pEYFP-N1 and pRb-pcDNA3.1 using the PEI reagent (Sigma-aldich Chemie GmbH, Steinheim, Germany) described previously 
. Cells were treated with or without 80 µM Pep-7 24 h post-transfection, and lysed for 30 min in lysis buffer 48 h post-transfection. Lysate supernate prepared as above were incubated with 10 µl rabbit anti-pRb antibody or mouse anti-flag (negative control) antibody and washed protein G agarose beads (Amersham Biosciences, Little Chalfont, UK) overnight at 4°C under continuous agitation. Beads were subsequently washed four times with the lysis buffer. Bound proteins were eluted by boiling in a Laemmli sample buffer and subjected to western blot assay.
SiHa cells seeded in 6-well plates were treated without or with 80 µM Pep-7 for 24 h. The cells were then treated with 50 µg/ml cycloheximide for 0 to 12 h. The cell lysates collected at different time points were subjected to Western blotting assay as above. Band intensity was used to determine the proteins half-life 
siRNA Synthesis and Cell Transfection
The siRNAs were synthesized by Shanghai Gene Pharma Co., Ltd. (Shanghai, P.R. China), as follows, E7 siRNA (target sequence: GCTTCGGTTGTGCGT
, sense: 5′-GCUUCGGUUGUGCGUACAA dTdT-3′
, antisense: 5′-UUGUACGCACAACCGAAGC dTdT-3′
, pRb-1, target sequence: 5′-AAGTTTCATCTGTGGATGGAG-3′
; pRb- 2, target sequence: 5′-AATGGTTCACCTCGAACACCC-3′
; p107-1, sense: 5′-GCCGGUUACAGAGUAUUGUTT-3′
; antisense: 5′-ACAAUACUCUGUAACCGGCTT-3′
; p107-2, sense: 5′-GCCCUUCUAAGAGUUUGAATT-3′
; antisense: 5′-UUCAAACUCUUAGAAGGGCTT-3′
; p130-1, sense: 5′-GGACUUAGUUUAUGGAAAUTT-3′
; antisense: 5′-AUUUCCAUAAACUAAGUCCTT-3′
; p130-2, sense: 5′-GCCCUUCUAAGAGUUUGAATT-3′
; antisense: 5′-AUUCUCAACAAAUAGCCGCTT-3′
; negative control sense: 5′-UUCUCCGAACGUGUCACGUTT-3′
, antisense: 5′-ACGUGACACGUUCGGAGAATT-3′ 
The cells were seeded in 12-well plate at a density of 3×105 cells/well. The following day, cells were transfected with SiRNA and luciferase reporter plasmids or siRNA alone, respectively, using LipofectAMINE 2000 as described in the manufacturer's protocol (Invitrogen, Carlsbad, CA, USA).
Luciferase Gene Reporter Assay
The reporter assays were performed using Bright-Glo™ Luciferase Assay System (Promega, Madison, WI, USA) according to the manufacturer's instructions. Briefly, cells were plated in 12-well plates at a density of 1.5×105 cells/well. The following day, cells were transfected with reporter constructs. After 24 h, cells were pre-treated with or without 80 µM Pep-7 for another 24 h. Thereafter, cell lysates were collected for reporter assays. The luciferase reporter activity of each sample was normalized against the corresponding protein content prior to calculation of the fold induction value relative to the controls. Each sample was determined in triplicate. The results represent means ± SE from three experiment runs.
RNA Extraction and qRT–PCR
Total RNA was extracted from the cultured cells using TRIzol reagent (Invitrogen, Carlsbad, CA,USA) according to the manufacturer's instructions. qRT-PCR was used to confirm the expression levels of mRNAs. Reverse transcription and qPCR were performed according to the protocol of Reverse Transcriptase System™ and SYBR® premix Ex Taq™ (Perfect Real Time) (TaKaRa, Dalian, P.R. China) on the ABI 7300 Real Time PCR System (Applied Biosystems, Foster City, CA, USA) supplied with analytical software. β-actin mRNA levels were used for normalization. Each sample was determined in triplicate. The results represent means ± SE from three experiment runs. The primers used for real-time PCR 
are listed in .
Primer sequences for real-time PCR.
Mice Maintenance and Subcutaneous Tumor Model
Female Balb/C-nu/nu mice were supplied by the Medical Experimental Animal Center of Guangdong Province (Guangdong, P.R. China). The care and use of laboratory animals have been approved by the Tsinghua University Animal Care and Use Committee, complying with the rules of REGULATIONS FOR THE ADMINISTRATION OF AFFAIRS CONCERNING EX-PERIMENTAL ANIMALS (Approved by the State Council of P.R. China)
A subcutaneous tumor model of nude mice was constructed. In brief, six-week-old female nude mice (18±2 g) were subcutaneously inoculated with 1.5×107
SiHa cells. When the tumor size reached about 100 mm2
, the mice were randomly divided into four groups with 5 mice each, namely, control group, low-dose group, middle-dose group, and high-dose group. The groups were treated once every other day for 16 days, with intravenous injections of PBS or Pep-7 at doses of 400 µg/mouse and 800 µg/mouse, respectively. The mice were raised for another 3 weeks. Tumor sizes were measured with a caliper every three days and tumor volume was calculated using “volume
/2.” At the end of the experiment, all mice were sacrificed and the weights of the detached tumors were measured. Tumor growth inhibition ratio was represented as the tumor weight relative to the control, calculated using the formula, “Relative tumor weight (% of control)
average tumor weight of treatment group/average tumor weight of control group×100%.”
Immunohistochemistry and H/E Staining
Three or two mice bearing tumor of each group were prelabeled with intra-peritoneal injection of BrdU (100 mg/kg, Sigma-Aldrich, Inc., Aaint Louis, MO, USA) 15 hours before sacrificed. Tumor specimens were then prepared as formalin-fixed, paraffin-embedded sections for hematoxylin-eosin staining, BrdU and pRb immunohistochemical staining as previously described 
. In brief, immunostaining was performed on formalin-fixed, paraffin-embedded sections using the anti-pRb monoclonal and anti- BrdU monoclonal antibodies, respectively. Before the application of the primary antibodies, an antigen retrieval technique was performed. For pRb assay, the deparaffinized and rehydrated slides were placed in 10 mmol/L citrate buffer (pH 6.0) and heated in a microwave oven for 15 min at 700 W. H2
was used to inhibit endogenous alkaline phosphatase. For BrdU incorporation test, sections were pretreated for 20 min with 0.1% trypsin in PBS at 37°C following the 30 min incubation in 2 N HCl at 37°C for DNA denaturation. After blocked, the slides were then incubated with anti-pRb or anti-BrdU monoclonal antibodies overnight at 4°C under dilutions of 1
100 and 1
500, respectively. The antibodies were detected by means of the Strept Avidin-Biotin Complex (SABC) method (Wuhan Boster Biological Technology, Ltd., Wuhan, P.R. China.) according to the manufacturer's instructions. Stained slides were examined under a Nikon ECLIPSE TE2000-U microscope (Nikon Corporation, Tokyo, Japan) and images were collected and analyzed with Pixera Penguin 600CL DiRactor™ (Pixera Corporation, Los Gatos, CA, USA). BrdU or pRb-positive cells were counted in each section. The total cell number was determined by nuclear staining with hematoxylin. At least 1000 total cells for each case were counted.
Statistics and Data Analysis
All cell culture-based experiments were repeated at least three times unless otherwise indicated. Images of colony formation, cell cycle distribution, cell apoptosis, Western blot, and immunostaining results from representative experiments are presented. The figures were created using Adobe Photoshop® CS graphics program. All data were analyzed by a paired t-test using SPSS 11.0 software. Differences were considered statistically significant at P<0.02.