C57Bl/6 p53 deficient female mice, TSG-p53N12-M, 5–6 weeks old, were purchased from Taconic Transgenics. All animals were housed in the University of Pittsburgh Animal Facility.
Immunohistochemical staining of cyclin B1 in tumor sections
Sections from mice were fixed in formalin (Thermo Fisher) for 24 hours and then embedded and sectioned (3–5µm). After drying overnight at 37°C, samples were deparaffinized, dehydrated, and stained with anti-cyclin B1 antibody (BD Biosciences) in the Pathology Laboratory, University of Pittsburgh. Briefly, samples were blocked with 2.5% BSA (Sigma) for 30 min at room temperature and stained with anti-cyclin B1 antibody. The avidin-biotin peroxidase method was then applied according to manufacturer’s protocol using the Vectastain ABC Elite staining kit (Vector laboratories, Inc., Burlingame, CA).
Construction of mouse and human cyclin B1 pcDNA3.1 DNA vectors
Human cyclin B1 cDNA derived from the HeLa cell line was a gift from Dr. Qimin Zhan at the University of Pittsburgh. Mouse cyclin B1 cDNA was an RT-PCR product derived from the mouse p53−/− cell line. Briefly, RT-PCR was performed using primers ATGGCGCTCAGGGTCACTAG (forward) and CAGTCTATTGGAGTTATGCCTTTG (reverse). A band at approximately 1.3kbp migrated on a 1.2% E-Gel Agarose gel (Invitrogen). The mouse cyclin B1 band was eluted using a MiniElute Kit (Qiagen, Valencia, CA) and subcloned into PCR2.1-TOPO vector (Invitrogen) and used to transform One-Shot TOP10 competent cells (Invitrogen) as described by the manufacturer. Colonies were picked for culture, and plasmids were isolated and identified positively by an EcoRI digest. Both cDNAs were then subcloned into the BamHI-XhoI site of the pcDNA3.1 expression vector (Invitrogen). All inserts were verified by DNA sequencing.
Synthesis of mouse cyclin B1 recombinant protein
Mouse cyclin B1 was a generous gift from IOMAI Corporation and was synthesized as described previously (Vella, et al, submitted). Briefly, pDEST-17 (Invitrogen) vector containing mouse or human cyclin B1 cDNA was used to transform Bl21 codon+ BL21 RIPL (Invitrogen) bacteria. Cyclin B1 was purified from inclusion bodies by extraction with guanidine-HCL under reducing conditions and passage over a Q-Sepharose FF (Amersham Biosciences) packed column to be collected in flow through fractions. Fractions were run over Nickel-HP 5 ml HighTrap columns (Amersham Biosciences) eluted in an imidazole gradient, and fractions collected and run on a gel to determine which ones contained the full-length protein. Protein purity was analyzed by coomassie blue, Western blot, HPLC, and Limulus amebocyte lysate assay (LAL, for endotoxin assessment).
DNA prime-protein boost vaccination and tumor measurements
At 6 weeks of age, experimental groups (n=15) were immunized with either pcDNA3.1 vector carrying mouse cyclin B1 cDNA, with the pcDNA3.1 empty vector, or left untreated. Mice were shaved on the abdomen with a No. 40 clipper 24 hours prior to treatments. The cDNA (4µg) was coated on 1–3µm gold particles (Bio-Rad, Hercules, CA) and fired into the shaved abdominal skin using a helium-powered gene gun. Three weeks later, the cyclin B1 immunized mice were boosted with protein in the presence of heat-labile enterotoxin (LT) placed on an immunostimulatory (IS) patch (IOMAI Corporation). Briefly, 25µg/100µl/mouse of mouse cyclin B1 recombinant protein was injected subcutaneously, with PBS injections used as controls in the adjuvant only group. In order to prevent grooming during the immunization procedure, mice were anesthetized intramuscularly with 100mg/kg ketamine (Phoenix Scientific Inc., St. Joseph, MO) mixed with 11mg/kg xylazine (Phoenix Scientific). The shaved skin was hydrated by rubbing with saline-drenched gauze. Hydrated skin was lightly blotted with dry gauze prior to immunization. The skin was abraded with sandpaper using gentle pressure 10 times in one direction. The area was then blotted with saline-saturated gauze and blotted dry. An immunostimulatory (IS) patch containing 20µg of heat-labile enterotoxin (LT) was applied to the treated skin. Mice were monitored for survival, the development visible tumors more than 2cm in diameter, or distress that met IACUC standards for euthanasia.
Measurement of Anti-Cyclin B1 Antibody Responses
Sera were drawn 3 weeks after the final immunization and tested for anti-cyclin B1 IgG by ELISA. Wells of 96-well ELISA plates (Thermo, Milford, MA) were each coated with 0.6µg recombinant mouse cyclin B1 protein in 50µl PBS. Plates were sealed overnight at 4°C and washed with PBS before use. Cyclin B1-coated wells and empty, background control wells were then blocked with 2.5% BSA in PBS (blocking buffer) for 1 hour. Plasma samples were diluted 1:100 in blocking buffer in 96-well polypropylene plates (Nunc, Thermo Fisher, Rockford, IL). 50µl of each diluted sample was then transferred to the ELISA plates. Samples were incubated for 1 hr and were subsequently washed with 1% PBS-Tween. Anti-mouse IgG (Sigma, St. Louis, MO) was diluted in blocking buffer and incubated on the plates for 1 hour. Plates were then washed as before and incubated substrate (TMB, BD Biosciences) for 30 minutes in the dark. The reaction was stopped with 2N H2SO4 and plates were read immediately at 450nm.