The E1B55kD gene deleted oncolytic adenovirus vector pAdel55 was established by nested PCR using pXC1 (Microbix Biosystems, Ontario, Canada) as the template. The viral region comprising nucleotides 1318–2038 was amplified using a primer set of 5 GCC GAC ATC ACC TGT GTC TAG AGA ATG -3′ (L1) and 5′- TCA GAT GGG TTT CTT CAC TCC ATT TAT CCT-3′ (R1). The region containing nucleotides 2005–2266 was amplified with another primer set of 5′-ATA AAG GAT AAA TGG AGT GAA GAA ACC CAT CTG AG-3′ (L2) and 5′-GAA GAT CTA TAC AGT TAA GCC ACC TAT ACA ACA-3′ (R2). Using the mixture of the two PCR products as template, a 955 bp fragment was then amplified using primers L1 and R2. This fragment was cut by Xba I and Bgl II and cloned into pXC1 to generate the plasmid pXC1-del55. SV40 polyA (160 bp) were obtained by PCR using pcDNA3 (invitrogen) as template and two primers: 5′-TGT GGA TCC TCT AGA GCT CGC TGA-3′ and 5′-TCT AGA TCT CGA GCC CCA GCT GGT-3′. Then it was digested with BamH I and Bgl II and cloned into the Bgl II site of pXC1- del55 to generate the plasmid pAdel55. The correct construction of this vector was confirmed by DNA sequencing. Constitutively active heat shock transcription factor 1 (cHSF1) and HSF1 miRNA inhibitor (HSF1i) gene were made as described previously . First, cHSF1, HSF1i, HSP70 or HSP90 gene was cloned into the polycloning site of shuttle vector pCA13 (Microbix Biosystems, Ontario, Canada). Then the whole gene expression cassette containing CMV promoter, transgene and SV40 polyA site was cut from pCA13-cHSF1, pCA13-HSF1i, pCA13-HSP70 or pCA13-HSP90 by Bgl II and subcloned into the corresponding site of pAdel55 to generate pAdel55-cHSF1, pAdel55-HSF1i, pAdel55-HSP70 or pAdel55-HSP90.
Adenovirus was generated by standard homologous recombination techniques using the plasmid pAdel55, pAdel55-cHSF1, pAdel55-HSF1i, pAdel55-HSP70 or pAdel55-HSP90 and the adenovirus packaging plasmid pBHGE3 (adenovirus packaging plasmid, Microbix Biosystems, Ontario, Canada) in HEK293 cells. Recombinant adenovirus plaque purified, propagated on HEK293 cells and purified by CsCl gradient according to standard techniques. Particle titers of all adenoviruses were determined by absorbance measurements at 260 nm, and functional plaque formation unit (pfu) titers were determined by plaque assay on HEK293 cells. The titer of stocks was 2.3 × 1011 pfu/ml for Adel55 (bioactivity 25.1), 5.1 × 1011 pfu/ml (bioactivity 23.5) for Adel55-cHSF1, 3.9 × 1011 pfu/ml (bioactivity 22.8) for Adel55-HSF1i, 2.9 × 1011 pfu/ml (bioactivity 23.3) for Adel55-HSP70, and 4.1 × 1011 pfu/ml (bioactivity 24.3) for Adel55-HSP90.
Cell lines and culture
C57B/6 mouse origin (H-2b) melanoma cell line B16 and Balb/C origin (H-2D) colorectal tumor cell line CT26 were obtained from American Type Tissue Collection (ATCC, Rockville, MD, USA). Cells were kept at 37 degree, 5% CO2 and 95% humidity in Dulbecco’s modified eagle medium (Cellgro, Herndon, VA, USA) supplemented with 10% (v/v) heat inactivated fetal bovine serum (Invitrogen, Carlsbad, CA, USA), 2 mM L-glutamine and 100 units/ml penicillin and 1,000 μg/ml streptomycin (Invitrogen, Carlsbad, CA, USA).
All animal experiments were carried out in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals. Five-week-old female C57B/6 or Balb/C mice were injected intradermally with 1 × 106 B16 or CT26 cells in a total volume of 50 μl by aseptic technique on the murine dorsum after shaving in sterile phosphate buffered saline. Tumor growth was measured with calipers. The perpendicular tumor diameter was measured every 5 days and tumor volume (V) was calculated by the formula for a rotational ellipsoid: V (mm3) = length × width2/2. In our multifocal tumor model, an initial inoculum was given on the left dorsum. On day 5, a secondary inoculum of tumor cells was transplanted on the opposite flank.
Animals were injected with 5 × 108 pfu Adel55, Adel55-cHSF1, Adel55-HSF1i, Adel55-HSP70, Adel55-HSP90 or phosphate buffered saline once a day for 2 consecutive days. Tumors were surgically excised 72 h later with small margins and the skin sutured using 3.0 nylon.
Cytotoxicity was assessed by the ability of spleen effector cells to lyse various tumor target cells. Splenocytes were derived by mechanical disruption of spleens under aseptic conditions in PBS. Red blood cells were removed by 5-min incubation in ammonium chloride lysis buffer (Pharmingen). Splenocytes were stimulated for 6 days in 24-well tissue culture plates at a ratio of 150:1 with 25 μg/ml mitomycin C-treated homolog cells in RPMI medium containing 10% fetal calf serum (RPMI-10% FCS) supplemented with 1 ng/ml recombinant murine IL-2 (BD Biosciences). Splenocytes were harvested from plates by Ficoll density centrifugation and added to 96- well U-bottom plates (Corning) in RPMI- 10% FCS. Target cells were harvested, washed in PBS and labeled with 200 uCi of 51Cr (Na2CrO4 in sterile saline; Amersham Biochemicals) in DMEM-10% FCS supplemented with 50 μM 2-mercaptoethanol for 90 min. Target cells were washed three times with ten times the volume of PBS and then added to plates containing splenocytes at the ratios described in the figure legends. After 4 h incubation at 37 degree, 5% CO2, the plates were harvested using a Skatron Harvesting System (Skatron). Chromium release into supernatant was counted using an ICN gamma counter. Spontaneous Cr leakage was measured by six wells that did not contain splenic effector cells. Maximum Cr leakage was determined by addition of Triton X-100 to a final concentration of 0.8% to six wells. Corrected % lysis was determined by the following formula: Corrected lysis = 100 × (effector cell sample chromium release - spontaneous target chromium released)/(maximum target chromium released - spontaneous target chromium released).
Data are expressed as means ± SD values. Student’s t test was applied to study the relationship between the different variables. Statistical significance was taken at P < 0.05. Tumor resistance statistics was determined by Fisher exact analysis. Survival analysis was performed using the Kaplan-Meier test.