Page 37 - 76_03

VOL. 76 (3), 327-342, 2010 ANTITUMORAL ACTIVITY OF ONCOLYTIC VACCINIA VIRUS...

The Western Reserve strain of vaccinia virus was used to generate
the recombinant viruses expressing the WT and the human mutant
PKR proteins. The recombinant genes were inserted in the TK locus
of viral genome. PKR expression is regulated by the lac I repressor
gene that is under the control of VACV early-late promoter p7.5. The
mutant PKR, K296R has lost the catalityc activity of PKR due to sub-
stitution of the lysine 296 by arginine. The mutant VACV viruses ex-
pressing PKR (VV-PKR), its inactive mutant form (VV-K296R) and lu-
ciferase (VVLUC) have been previously described (9, 10). Viruses were
grown in monkey BSC-40 cells and purified by sucrose gradients.

TRAMP-C1 cells grown in12-well plates were infected at low 0.01
pfu/cell or high 5 pfu/cell virus multiplicities. Following virus adsorp-
tion for 60 min at 37 ºC, the inoculum was removed and cells incubat-
ed with fresh DMEM containing 2% FCS at 37 ºC in a 5% CO2 atmos-
phere. At different times postinfection, cells were collected by scraping
and used either for Western blot or virus titration. At 24 h postinfec-
tion (hpi), infected cells were lysed in Laemmli buffer, cell extracts frac-
tionated by 12% SDS-PAGE and analyzed by Western blot using rab-
bit polyclonal anti-vaccinia serum, anti-PKR or anti-phospho PKR.
Virus yields were determined by plaque formation at 48 hpi in mon-
key BSC-40 cells after staining with 1% crystal violet in 2% ethanol.

2.2. Evaluation of virus pathogenicity

Mice C57/BL6, four per group, were inoculated at days 0 and 3 with
a dose of 5x107 pfu per mouse with different viral recombinants,
VVLUC, VV-K296R or VV-PKR, by either intraperitoneal or intranasal
routes. Mice were weighed daily and signs of illness were scored. Virus
was titrated from homogenates of spleen and lung of each mouse at
different days post-infection by a plaque assay in monkey BSC-40 cells.

2.3. Evaluation of oncolytic activity

Male mice C57/BL6 seven weeks old, 10 animals per group, were
injected by subcutaneous route with 1x106 of TRAMP-C1 of mouse
prostate cancer cells. At the median tumour volume of 75 to 100 mm3

331